recombinant hcg: state-of -art formulation for a patient-centered management in art

Sandro C. Esteves, MD, PhD Medical Director, ANDROFERT

Campinas, Brazil

Recombinant hCG

MerckSeromo Symposium, Singapore 2014

The state of the art formulation for a patient-centered management in ART

Learning objectives At the completion of this presentation, participants should be able to: •  Review the role of hCG in IVF •  Understand the differences between urinary

and recombinant hCG •  Learn the clinical advantages of using rec-

hCG for oocyte maturation triggering

ANDROFERT androfert.com.br

ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 2 2014 SEPTEMBER

ANDROFERT

Human Chorionic Gonadotropin •  Glycoprotein produced

during pregnancy by syncytiotrophoblast cells and by the pituitary in menopause women

•  In early pregnancy, hCG rescues the corpus luteum and maintains progesterone production until placental steroidogenesis is established



ANDROFERT

Extracellular fluid

Cytoplasm

Plasma membrane

LH hCG

LH/hCG receptor



ANDROFERT

Sharing the same α subunit and 81% of the

aminoacid residues of the β subunit, LH and hCG bind to the same

receptor


ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 5 2014 September

ANDROFERT

Beta unit

Carboxyl terminal segment

Longer in hCG Higher

receptor affinity in hCG

Absent in LH and present

in hCG Longer half-life in

hCG

Sources of LH Activity

hCG

LH

Leao & Esteves. Clinics 2014; 69(4): 279–293.

LH hCG No. AA beta subunit 121 145 Receptor binding affinity Low High* No. glycosylation sites1 1 6 Initial half-life (h) 0.6-1.3 3.9-5.5 Terminal half-life (h) 9-12 23-31 Bioequivalency 6 – 8 IU 1 IU

1N-linked and O-linked glycosylation sites in beta subunit *2-3x higher than LH

LH and hCG not identical “sisters”

Leao & Esteves. Clinics 2014; Choi & Smitz. Mol Cell Endocrinol20142014



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14h

14h 20h

48h 0 20 h

Natural LH surge

hCG

Adapted from Chan et al. Hum Reprod. 2003;18:2294-7



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5 days

In IVF, hCG administration has been the gold standard for final follicular maturation as a surrogate for the mid-cycle LH surge

hCG and IVF - History •  Zondek & Ascheim (1927)

found that urine of pregnant women contained a substance capable of inducing follicular maturation and ovarian stromal luteinization

•  First hCG preparations developed in 1931 from urine of pregnant women



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Urinary hCG production The source: •  Pooled urine from pregnant

women The process: •  Urine pool is processed to

concentrate gonadotropins •  hCG is extracted and purified

by either antibody affinity column or conventional chromatography



ANDROFERT Source: Dr. Shelley Slaughter, FDA

Randomized 21-22 day old female rats

hCG injected sc 1x for 3 days

Sacrifice day 4 and collect ovaries

Ovaries are weighed and

data processed

Quantification of hCG activity based on rat ovarian weight gain



ANDROFERT Steelman & Pohley Endocrinol. 53:604-16, 1953

Proportion of total immunoreactivity (%)

Pregnyl® Choragon® Profasi®

Intact bioactive hCG 50 30 96 Hyperglycosylated hCG 0.6 4 0.5 Free β subunit 6.2 8 2.4 β-core fragment1 43 58 1.2 Epidermal growth factor2 181-204 154 4-10

Yarram et al. Fertil Steril 2004;82:232-3

1degradation product of hCG; 2EGF is a contaminant (ng/5000IU)

Level of functionally intact hCG and contamination among formulations



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Non-gonadotropin proteins identified in u-hCG products

PLoS ONE 2011; 6(3): e17815

What is the ideal gonadotropin preparation?

•  Originating from a safe and consistent source •  Manufactured using consistent and

standardized processes •  Fully physiochemically characterized •  Effective •  Favorable safety profile •  Patient-convenient



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2001: rec-hCG (Choriogonadotropin alfa)

Adapted from Leao & Esteves. Clinics 2014;69:279-93



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Secretion of hCG molecules

Incorporation into host cell chromosome

Bulk hCG Released

Bulk hCG



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Purity (hCG content)

Specific activity (hCG/mg protein)1,2

Protein/IU (mcg = IU)

u-hCG <70% ~13,500 IU/mg ~550 = 5000

rec-hCG 99.9% 27,000 IU/mg 250 = 6750

1Bassett R et al Current Med Res Opinion 2005, 21:1969-76; 2Stenman U-H et al Br

J Pharmacol 2008; 154:569-83; Van Dorsselaer A et al PLoS One. 2011;6:e17815

u- hCG rec- hCG

CG beta CG alpha

Contaminants



ANDROFERT

Prion protein peptides in hCG preparations analyzed by Liquid chromatography

selected-reaction monitoring (LC-SRM)

Van Dorssealer et al. PLoS ONE 2011; 6(3): e17815



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Size Exclusion High Performance Liquid Chromatography (SE- HPLC)

Gervais et al. Glycobiology 2003;13:179-189; Leao & Esteves Clinics 2014;69:279-93.

Filled by Mass Approach

Proven consistency of rec-hCG physiochemical characteristics

Consistent specific activity i.e. the same biological activity measured in IU/mg of protein

Approval to fill (vials, pens) by mass

(protein content in solution)



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rec- hCG

u- hCG: lyophilized powder to be reconstituted



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Rec-hCG has higher purity and better safety profile compared with u-hCG

Consistent physiochemical characteristics allow rec-hCG to be filled by mass, thus offering better dose precision and low batch-to-batch variation compared with u-hCG (filled by bioassay)

High specific activity of rec-hCG allows SC administration using a pre-filled syringe or a pen device while u-hCG needs to be reconstituted and is primarily indicated for IM route

Differences between urinary and recombinant hCG

Key points



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Clinical advantages of

rec-hCG in IVF as used for oocyte maturation triggering

Approved for use in the treatment of: •  Adult women undergoing superovulation

prior to ART such as IVF to trigger final follicular maturation and luteinization after stimulation of follicular growth;

•  Anovulatory of oligo-ovulatory women to trigger ovulation and luteinization after stimulation of follicular growth.



ANDROFERT

250 μg rhCG=6,500 IU; SC

10,000 IU uhCG; IM

5,000 IU uhCG; IM

Seru

m h

CG

leve

ls

Trinchard-Lugan et al., 2002 Itskovitz et al., 1991



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0% 20% 40% 60% 80% 100%

Doctors & embryologists

Nurses

Patients Safety

Effectiveness

Patient-centeredness

Hum Reprod 2013;26(6):1584-97



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r-hCG (250 mcg)

r-hCG (500 mcg)

u-hCG (10,000 IU)

No. patients 94 89 92 Oocytes retrieved (mean) 13.6 14.6 13.7 2PN fertilized (mean) 7.2 8.8 P=0.02 7.8 Serum P (d6-7 after hCG) 133.0 163.5 P=0.03 147.4 Pregnancy rate (%) 35.1 36 35.9 OHSS (%) 3.2 9.0 3.1

Chang et al. Fertil Steril. 2001; 76: 67–74



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RCT N Effect Oocytes retrieved 9 1409 MD: -0.04 (95% CI -0.69 to 0.61)

Live birth 6 1,019 OR: 1.04 (95% CI 0.79 to 1.37)

Miscarriage 7 1,106 OR: 0.69 (95% CI 0.41 to 1.18)

Severe OHSS 3 549 OR: 1.49 (95% CI 0.54 to 4.1)

Youssef et al. Cochrane Database Syst Rev. 2011; 13(4):CD003719.



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Databases searched up to January 2010

Farrag et al. JARG 2008; 25:461-6.



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8.4 7.3 7.1 4.7

0 2 4 6 8

10

No. Retrieved oocytes No. MII with mature cytoplasm

rec-hCG (250 mcg; n=42)

u-hCG (10,000 IU; n=47)

*p<0.01 *

Effectiveness RCT comparing rec-hCG (250 mcg) with u-hCG (10,000 IU) for oocyte maturation triggering on

delivery rates in eSET antagonist cycles

26.7% 44.1%

Delivery rate (%)

10,000 IU u-hCG 250 mcg rec-hCG

N=119 aged<32

OR: 2.16 (95% CI: 1.01-4.67; p=0.04) Papanikolaou EG et al. Fertil Steril 2010; 94:2902-4



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Saleh L et al. Placenta. 2007;28(2-3):199-203

•  In vitro treatment of trophoblast cells with different hCG products

•  Phosporilation of EGC receptors after u-hCG treatment, but not with rec-hCG

•  Increased trophoblast invasion and syncytialization observed using EGF-free hCG (rec-hCG) compared with u-hCG (EGF-contaminated)

Urinary and rec-hCG differentially impact trophoblast differentiation



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RCT N Odds-ratio

Local site reactions* rec-hCG vs. u-hCG 3 374 0.39

95% CI: 0.25 to 0.61

Driscoll et al. 2000: 27% vs 42% ERHCG Group 2000: 23% vs 45% Abdelmassih et al. 2005: 23% vs 45%

Youssef et al. Cochrane Database Syst Rev. 2011; 13(4):CD003719.



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* Pain and/or inflammation

Human errors after hCG administration

•  0.5%-0.8% Empty follicle syndrome after u-hCG administration1,2

•  Case report of patient who injected only diluent3

•  Recurrent empty follicle syndrome successfully treated with rec-hCG4

1Quintans et al. Hum Reprod 1998;13:2703-5; 2Zegers Hochschild et al. Hum Reprod 1995;10:2262-5; 3Esposito & Patrizio J Reprod Med 2000;45:511-4;

4Penarrubia et al. Hum Reprod 1999;14:1703-6.



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Serum hCG after subcutaneous administration of 250 mcg rec-hCG in 473

patients undergoing IVF No. retrieved

oocytes hCG levels (mUI/mL);

Mean ± SD 0 103.3 ± 35.5

1-5 121.9 ± 51.9 6-10 119.5 ± 48.0 11-15 116.0 ± 39.1 >15 125.7 ± 43.6

Matorras et al. JARG 2012; 29:1067–71



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hCG preferences in treatment-experienced patients at Androfert

Total (n=76) 60% 29%

3%

8%

prefer new pen prefer pre-filled syringe prefer lyophilized powder to reconstitute Not matter



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22 studies sampling 21,453 patients from eight countries



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•  Safety originates from a safe and consistent contamination-free source; fully characterized

•  Effectiveness 250 mcg rec-hCG at least as effective as 10,000 IU u-hCG for final follicular maturation

•  Patient-centeredness better tolerated and preferred by treatment-experienced patients

Clinical advantages of rec-hCG in IVF for oocyte maturation triggering

Key points



ANDROFERT

recombinant hcg: state-of -art formulation for a patient-centered management in art

Health & Medicine

male reproductions esteves

hcg administration

quantification of hcg

role of hcg

lhand hcg bindto

urinaryand recombinant

intact hcg andcontamination

sources of lh