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Analytical and Biological Characterization of a Noninnovator Insulin Glargine
and the Originator Drug Product
Article in Journal of Diabetes Science and Technology · October 2015
DOI: 10.1177/1932296815606914
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Abraham Escobedo-Moratilla Francisco Kuri-Brena
Health Affairs Consulting SAPI de CV Landsteiner Scientific, Mexico City
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Jose Perez-Urizar Ana Barba de la Rosa
Autonomous University of San Luis Potosí Instituto Potosino de Investigación Científica y Tecnológica
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DSTXXX10.1177/1932296815606914Journal of Diabetes Science and TechnologyEscobedo-Moratilla et al
Letter to Editor
Journal of Diabetes Science and Technology
Analytical and Biological Characterization
1–2
© 2015 Diabetes Technology Society
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of a Noninnovator Insulin Glargine and sagepub.com/journalsPermissions.nav
DOI: 10.1177/1932296815606914
the Originator Drug Product dst.sagepub.com
Abraham Escobedo-Moratilla, MSc1, Francisco Kuri-Breña Romero de Terreros, PhD2,
José Pérez-Urizar, PhD3, and Ana Paulina Barba de la Rosa, PhD1
Keywords
analytical characterization, biosimilar, biotechnology, insulin analogues, mass spectrometry, protein structure
In Mexico, the Federal Commission for Prevention of both LNT and BNG products. Some differences in secondary
Sanitary Risks (COFEPRIS) approved in 2000 the use of structure were found; however, all other properties had compa-
insulin glargine for glycemic control in adults and children rable results within analyzed batches of LNT and BNG.
with type 1 diabetes mellitus and in adults with type 2 diabe- Biological activity performed in rabbits and based in the USP
tes mellitus. assay demonstrated no statistical differences between formula-
Insulin glargine is a long-acting insulin analog, which has tions. These findings suggest that slight structural differences
53 amino acids with a molecular weight (MW) of 6063.0 Da between formulations do not have a biological activity impact.
and is produced in Escherichia coli K12 strains. This analog In Mexican and international regulatory guidelines,3,4 a
is constituted by A- and B-chain with 21 and 32 amino acids, noninnovator biopharmaceutical can be considered a bio-
respectively, linked by 2-disulfide bonds.1 similar product if it complies with comparable properties at a
To develop cost-effective therapies, several manufactur- structural level, as well as clinical efficacy and safety, so this
ers have been launched to the market biopharmaceuticals is the first step in the regulatory pathway of BNG as an insu-
containing the same active principle as innovator products lin glargine proposed as biosimilar. It was not expected that
whose patent has been expired; these noninnovator products BNG would be identical to LNT, because although they are
are considered biosimilar drugs when comparable efficacy made with the same E. coli strain, they are generated with a
and safety to the innovator product are demonstrated through different manufacturing process, so the final product will be
structural and physicochemical assessment, as well as pre- similar but not identical.2,4,5 However, different emphasis in
clinical and clinical studies.2 regulation of biosimilar insulins is present across the world
In Mexico, two insulin glargine formulations are com- because each country (or continent in the case of European
mercially available: the innovator product, Lantus® (LNT), Union) has its own criteria;6 in the case of Mexico, although
manufactured by Sanofi-Aventis (DF, Mexico), and a nonin- regulation is now harmonized with the international guide-
novator version, Bonglixan® (BNG), which is manufactured lines4 to provide effective and safe therapies to society, the
and distributed in Mexico by Landsteiner Scientific (Toluca, guidelines are still broad compared with the European Union
Mexico). BNG was registered before the biosimilar regula- and countries like the United States and Canada, which have
tory guideline was published in Mexico,3 therefore a lack of specific guidelines for insulins.6
information exist about its structural and physicochemical These results provide the basis to validate BNG as a pro-
properties, so here we report an analytical and biological posed biosimilar according to Mexican regulatory guidelines.
assessment to confirm the properties of BNG compared to
LNT as the originator product. Three batches of LNT and 1
IPICyT, Instituto Potosino de Investigación Científica y Tecnológica A.C.
two batches of BNG were used. The evaluation was divided Camino a la Presa San José 2055, San Luis Potosí, Mexico
2
into three stages: (1) analytical assessment, including reducing Landsteiner Scientific S.A. de C.V., Mexico
3
and nonreducing SDS-PAGE, western blot, ESI-Q-TOF-MS, Dixpertia, Investigación Biofarmacéutica y Farmacológica S.C., San Luis
Potosí, Mexico
peptide mapping by UPLC-MS, and RP-HPLC, (2) structural
analysis, including spectroscopic UV and CD and profiles, Corresponding Authors:
and (3) in vivo biological analysis in rabbits (see the Supple- Ana Paulina Barba de la Rosa, PhD, IPICyT, Instituto Potosino de
Investigación Científica y Tecnológica A.C. Camino a la Presa San José
mentary Methods and Information at https://drive.google.com/
2055, Col. Lomas 4ª Sección, C.P. 78216, San Luis Potosí, Mexico.
folderview?id=0BzcpakuIk-ajcjF6ZTZ2WGVOSFU&usp= Email: [email protected].
sharing). Table 1 summarizes the analytical and biological proper- José Pérez Urizar, PhD, Dixpertia.
ties determined with the corresponding method to characterize Email: [email protected]
Downloaded from dst.sagepub.com at CAMBRIDGE UNIV LIBRARY on October 2, 2015
2 Journal of Diabetes Science and Technology
Table 1. Summary of Analytical and Biological Properties Determined in the Study for Both Lantus and Bonglixan.
Properties Technique LNT1 LNT2 LNT3 BNG1 BNG2
Electrophoretic pattern SDS-PAGE One band in nonreducing conditions
Disulfide bonds SDS-PAGE Two bands in reducing conditions
Immunochemical reactivity WB Immunologic binding with antihuman insulin antibody
Ionization pattern ESI-Q-TOF-MS Three main ionization states: 1011 m/z (+6), 1213 m/z (+5) and 1516 m/z (+4)
Molecular weight (Da) ESI-Q-TOF-MS 6062.8003 6061.8403 6062.8003 6061.8403 6062.4404
Peptide mapping nanoUPLC-MS Detection of all the expected peptides according to the theoretical sequence
Chromatography pattern RP-HPLC Presence of one peak
Impurities RP-HPLC Absence of nonassociated peaks
Thermal degradation RP-HPLC Two degradation products: 0.58 and 0.73 min
CD spectroscopic pattern CD Homogeneity between batches of the same product, some differences between LNT and
BNG in the 190-195 and 205 nm regions.
Secondary structure (α-helix, CDSSTR method 0.51, 0.16 0.48, 0.19 0.31, 0.26 0.26, 0.24 0.25, 0.26
β-strand fractions)
UV spectroscopic pattern UV Homogeneity between batches of the same product, some differences between LNT and
BNG in the 240-255 nm regions
Biological activity USP-based Without statistical differences within analyzed batches
method
BNG, Bonglixan; LNT, Lantus. 1, 2, and 3 different batches analyzed.
Abbreviations Funding
BNG, Bonglixan; CD, circular dichroism; COFEPRIS, Federal The author(s) disclosed receipt of the following financial support
Commission for Prevention of Sanitary Risks; ESI, electrospray for the research, authorship, and/or publication of this article: AEM
ionization; LNT, Lantus; MS, mass spectrometry; Q-TOF, quadru- has a PhD scholarship fund (no 362391) provided by CONACYT,
pole-time of flight; RP-HPLC, reversed phase high performance Mexico. Work was supported by PROINNOVA-PEI, no 179521.
liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis; UPLC, ultra performance liquid References
chromatography; USP, United States Pharmacopeia; UV, ultravio- 1. European Medicines Agency. Scientific discussion for approval
let spectrophotometry; WB, western blot. of Lantus. 2003. Available at: http://www.ema.europa.eu/docs/
en_GB/document_library/EPAR_-_Scientific_Discussion/
Acknowledgments human/000284/WC500036075.pdf. Accessed October 10, 2014.
We thank for their technical assistance Alberto Barrera-Pacheco for 2. Dranitsaris G, Amir E, Dorward K. Biosimilars of biological
MS assays and Roberto Torres-Ramírez for the biological activity drug therapies: regulatory, clinical and commercial consider-
assay. We appreciate kindly the help provided in the CD analysis by ations. Drugs. 2011;71:1527-1536.
Laboratorio de Fisicoquímica e Ingeniería de Proteínas (UNAM, 3. DOF (Official Journal of the Federation). NOM-177-SSA1-2013.
Mexico). We also acknowledge CONACyT-Mexico grant 56787 2013. Available at: http://www.dof.gob.mx/nota_detalle.php?
(Laboratory for Nanoscience and Nanotechnology Research- codigo=5314833&fecha=20/09/2013. Accessed October 10, 2014.
LINAN) and INFRA-2013 no 204373. Thanks to Antonio 4. World Health Organization. 2009. Guidelines on evaluation of similar
Castellanos (CNS-IPICyT) for his valuable help and WATERS biotherapeutic products (SBP’s). Available at: http://www.who.int/
Mexico for their technical support. biologicals/areas/biological_therapeutics/BIOTHERAPEUTICS_
FOR_WEB_22APRIL2010.pdf. Accessed October 10, 2014.
5. Schiestl M, Stangler T, Torella C, Cepeljnik T, Toll H, Grau
Declaration of Conflicting Interests R. Acceptable changes in quality attributes of glycosylated bio-
The author(s) declared the following potential conflicts of interest pharmaceuticals. Nat Biotechnol. 2011;29(4):310-312.
with respect to the research, authorship, and/or publication of this 6. Heinemann L, Khatami H, McKinnon R, Home P. An overview
article: Drug formulations and partial financial support for the study of current regulatory requirements for approval of biosimilar
were provided by Landsteiner Scientific S.A. de C.V. insulins. Diabetes Technol Ther. 2015;17(7):510-526.
Downloaded from dst.sagepub.com at CAMBRIDGE UNIV LIBRARY on October 2, 2015
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