In-vivo biological activity and glycosylation analysis of a biosimilar recombinant human follicle-stimulating hormone product (Bemfola) compared with its reference medicinal product (GONAL-f)

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Abstract

Recombinant human follicle-stimulating hormone (r-hFSH) is widely used in fertility treatment. Although biosimilar versions of r-hFSH (follitropin alfa) are currently on the market, given their structural complexity and manufacturing process, it is important to thoroughly evaluate them in comparison with the reference product. This evaluation should focus on how they differ (e.g., active component molecular characteristics, impurities and potency), as this could be associated with clinical outcome. This study compared the site-specific glycosylation profile and batch-to-batch variability of the in-vivo bioactivity of Bemfola, a biosimilar follitropin alfa, with its reference medicinal product GONAL-f. The focus of this analysis was the site-specific glycosylation at asparagine (Asn) 52 of the α-subunit of FSH, owing to the pivotal role of Asn52 glycosylation in FSH receptor (FSHR) activation/signalling. Overall, Bemfola had bulkier glycan structures and greater sialylation than GONAL-f. The nominal specific activity for both Bemfola and GONAL-f is 13,636 IU/mg. Taking into account both the determined potency and the nominal amount the average specific activity of Bemfola was 14,522 IU/mg (105.6% of the nominal value), which was greater than the average specific activity observed for GONAL-f (13,159 IU/mg; 97.3% of the nominal value; p = 0.0048), although this was within the range stated in the product label. A higher batch-to-batch variability was also observed for Bemfola versus GONAL-f (coefficient of variation: 8.3% vs 5.8%). A different glycan profile was observed at Asn52 in Bemfola compared with GONAL-f (a lower proportion of bi-antennary structures [~53% vs ~77%], and a higher proportion of tri-antennary [~41% vs ~23%] and tetra-antennary structures [~5% vs <1%]). These differences in the Asn52 glycan profile might potentially lead to differences in FSHR activation. This, together with the greater bioactivity and higher batch-to-batch variability of Bemfola, could partly explain the reported differences in clinical outcomes. The clinical relevance of the differences observed between GONAL-f and Bemfola should be further investigated.

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Structural biology of glycoprotein hormones and their receptors: insights to signaling.

Xuliang Jiang, James Dias, Xiaolin He (2014)

This article reviews the progress made in the field of glycoprotein hormones (GPH) and their receptors (GPHR) by several groups of structural biologists including ourselves aiming to gain insight into GPH signaling mechanisms. The GPH family consists of four members, with follicle-stimulating hormone (FSH) being the prototypic member. GPH members belong to the cystine-knot growth factor superfamily, and their receptors (GPHR), possessing unusually large N-terminal ectodomains, belong to the G-protein coupled receptor Family A. GPHR ectodomains can be divided into two subdomains: a high-affinity hormone binding subdomain primarily centered on the N-terminus, and a second subdomain that is located on the C-terminal region of the ectodomain that is involved in signal specificity. The two subdomains unexpectedly form an integral structure comprised of leucine-rich repeats (LRRs). Following the structure determination of hCG in 1994, the field of FSH structural biology has progressively advanced. Initially, the FSH structure was determined in partially glycosylated free form in 2001, followed by a structure of FSH bound to a truncated FSHR ectodomain in 2005, and the structure of FSH bound to the entire ectodomain in 2012. Comparisons of the structures in three forms led a proposal of a two-step monomeric receptor activation mechanism. First, binding of FSH to the FSHR high-affinity hormone-binding subdomain induces a conformational change in the hormone to form a binding pocket that is specific for a sulfated-tyrosine found as sTyr 335 in FSHR. Subsequently, the sTyr is drawn into the newly formed binding pocket, producing a lever effect on a helical pivot whereby the docking sTyr provides as the 'pull & lift' force. The pivot helix is flanked by rigid LRRs and locked by two disulfide bonds on both sides: the hormone-binding subdomain on one side and the last short loop before the first transmembrane helix on the other side. The lift of the sTyr loop frees the tethered extracellular loops of the 7TM domain, thereby releasing a putative inhibitory influence of the ectodomain, ultimately leading to the activating conformation of the 7TM domain. Moreover, the data lead us to propose that FSHR exists as a trimer and to present an FSHR activation mechanism consistent with the observed trimeric crystal form. A trimeric receptor provides resolution of the enigmatic, but important, biological roles played by GPH residues that are removed from the primary FSH-binding site, as well as several important GPCR phenomena, including negative cooperativity and asymmetric activation. Further reflection pursuant to this review process revealed additional novel structural characteristics such as the identification of a 'seat' sequence in GPH. Together with the 'seatbelt', the 'seat' enables a common heteodimeric mode of association of the common α subunit non-covalently and non-specifically with each of the three different β subunits. Moreover, it was possible to establish a dimensional order that can be used to estimate LRR curvatures. A potential binding pocket for small molecular allosteric modulators in the FSHR 7TM domain has also been identified.

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What does it take to make a developmentally competent mammalian egg?

Maurizio Zuccotti, Valeria Merico, Sandra Cecconi … (2016)

A limitation to our ability to distinguish between developmentally competent and incompetent eggs is our still only partial knowledge of the critical features that are needed to make a good egg and when during oogenesis these specific characteristics are acquired. The main objective of this review is to summarize the results of areas of investigation that are contributing to our still inadequate understanding of the molecular aspects of making developmentally competent eggs. For each area discussed, a systematic search was made using PubMed. The search was without temporal limits but mainly yielded publications between 1982-1999 (23%) and 2000-2011 (77%). Taking an oocyte-centred view, we describe throughout folliculogenesis: (i) the factors that regulate oocyte growth; (ii) the role of oocyte-cumulus cell dialogue; (iii) the epigenetic organization of the oocyte genome and (iv) the storage and regulation of maternal RNAs. The multifaceted complex of factors involved in oocyte growth constitutes the backbone on which oocyte developmental competence is built up. Operating behind the expression of these factors is a specific epigenetic signature established during oogenesis, but our knowledge is only approximate and major efforts will be required for more accurate analyses at specific gene loci. The growing research on small silencing RNAs during oogenesis and early oocyte development is revealing these molecules' critical role in mRNA degradation. Our next challenge will be to dissect the complex interactions among the different molecular players identified and to establish the presence of functional links among these factors.

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Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 2: Mass spectrometric methods

Dietmar Reusch, Markus Haberger, David Falck … (2015)

To monitor the Fc glycosylation of therapeutic immunoglobulin G in bioprocess development, product characterization and release analytics, reliable techniques for glycosylation analysis are needed. Several analytical methods are suitable for this application. We recently presented results comparing detection methods for glycan analysis that are separation-based, but did not include mass spectrometry (MS). In the study reported here, we comprehensively compared MS-based methods for Fc glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods investigated were compared with respect to precision, accuracy, throughput and analysis time. Emphasis was put on the detection and quantitation of sialic acid-containing glycans. Eleven MS methods were compared to hydrophilic interaction liquid chromatography of 2-aminobenzamide labeled glycans with fluorescence detection, which served as a reference method and was also used in the first part of the study. The methods compared include electrospray MS of the heavy chain and Fc part after limited digestion, liquid chromatography MS of a tryptic digest, porous graphitized carbon chromatography MS of released glycans, electrospray MS of glycopeptides, as well as matrix assisted laser desorption ionization MS of glycans and glycopeptides. Most methods showed excellent precision and accuracy. Some differences were observed with regard to the detection and quantitation of low abundant glycan species like the sialylated glycans and the amount of artefacts due to in-source decay.

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Author and article information

Contributors

Renato Mastrangeli:

ORCID: http://orcid.org/0000-0002-0294-8773

Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing

Abhijeet Satwekar: Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing

Francesca Cutillo: Role: Formal analysisRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing

Cinzia Ciampolillo: Role: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – review & editing

Wolf Palinsky: Role: Writing – review & editing

Salvatore Longobardi: Role: ConceptualizationRole: Funding acquisitionRole: ResourcesRole: Writing – original draftRole: Writing – review & editing

Frederique Lisacek: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 7 September 2017

Publication date Collection: 2017

Volume: 12

Issue: 9

Electronic Location Identifier: e0184139

Affiliations

[1 ] Biotech Development Programme, Merck Serono S.p.A. (an affiliate of Merck KGaA, Darmstadt, Germany), Guidonia Montecelio, Rome, Italy

[2 ] Pharamceutical & Analytical Development Biotech Products, Merck Serono S.p.A. (an affiliate of Merck KGaA, Darmstadt, Germany), Guidonia Montecelio, Rome, Italy

[3 ] Analytical BQC, Merck RBM S.p.A. (an affiliate of Merck KGaA, Darmstadt, Germany), Ivrea, Turin, Italy

[4 ] Biotech Development Programme, Merck Biopharma (an affiliate of Merck KGaA, Darmstadt, Germany), Aubonne, Switzerland

[5 ] Global Medical Affairs Fertility, Merck Serono S.p.A. (an affiliate of Merck KGaA, Darmstadt, Germany), Guidonia Montecelio, Rome, Italy

Swiss Institute of Bioinformatics, SWITZERLAND

Author notes

Competing Interests: All authors are current employees of Merck Serono S.p.A., an affiliate of Merck KGaA, Darmstadt, Germany. This paper describes data for a product currently marketed by Merck KGaA, Darmstadt, Germany. This does not alter the authors’ adherence to all PLOS ONE policies on sharing data and materials.

* E-mail: renato.mastrangeli@ 123456merckgroup.com

Author information

Renato Mastrangeli http://orcid.org/0000-0002-0294-8773

Article

Publisher ID: PONE-D-16-44640

DOI: 10.1371/journal.pone.0184139

PMC ID: 5589168

PubMed ID: 28880909

SO-VID: 8f83befd-8bd0-4454-adb5-ef9cbfb84b19

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 24 December 2016

Date accepted : 18 August 2017