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Physicochemical and Biological Characterization of RTXM83, a New Rituximab Biosimilar

  • María L. Cerutti
  • Analía Pesce
  • Cédric Bès
  • Mauricio SeigelchiferEmail author
Original Research Article



RTXM83 is a rituximab biosimilar with proven clinical safety and efficacy. It is the first rituximab biosimilar developed and approved in South America and is currently marketed in several Latin American, Middle Eastern and African countries.


The aim of this study was to present the physicochemical and biological characterization studies utilized to demonstrate the similarity between RTXM83 and its reference product.


Primary and higher order protein structures were analysed using peptide mapping with liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS), fluorescence spectroscopy and circular dichroism, and micro-differential scanning calorimetry, among other techniques. Charge variants were determined by cation-exchange chromatography (CEX) and capillary isoelectric focusing (cIEF). Glycosylation and glycoforms distribution were analysed using MS, normal phase high-performance liquid chromatography (NP-HPLC) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Size variants were evaluated by size-exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation (SV-AUC), dynamic light scattering (DLS), and capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Biological characterization included binding assays for complement C1q, CD20, and several Fc receptors (FcRs), as well as potency determination for in vitro apoptosis induction, complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC).


RTXM83 and the reference product showed identical primary sequences and disulfide bridge patterns, and similarity at higher order protein structures, post-translational modification profiles (amino acid modifications, charge variants, and glycosylation) and levels of purity and process-related impurities. Functional studies demonstrated that RTXM83 is similar to the reference product regarding the three known mechanisms of action of rituximab: CDC, ADCC, and apoptosis induction. Binding affinities to CD20, complement component C1q, and different FcRs were also equivalent.


RTXM83 is similar to its reference product in all critical quality attributes.


Compliance with Ethical Standards


This study was sponsored by Mabxience.

Conflict of interest

María Laura Cerutti, Analía Pesce, Cédric Bès and Mauricio Seigelchifer are current or previous employees of Mabxience. They declare that they have no other conflicts of interest.

Supplementary material

40259_2019_349_MOESM1_ESM.pptx (5.1 mb)
Supplementary material 1 (PPTX 5241 kb)


  1. 1.
    Reff ME, Carner K, Chambers KS, Chinn PC, Leonard JE, Raab R, et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood. 1994;83(2):435–45.Google Scholar
  2. 2.
    Maloney DG, Grillo-Lopez AJ, White CA, Bodkin D, Schilder RJ, Neidhart JA, et al. IDEC-C2B8 (rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin’s lymphoma. Blood. 1997;90(6):2188–95.Google Scholar
  3. 3.
    Maloney DG, Liles TM, Czerwinski DK, Waldichuk C, Rosenberg J, Grillo-Lopez A, et al. Phase I clinical trial using escalating single-dose infusion of chimeric anti-CD20 monoclonal antibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma. Blood. 1994;84(8):2457–66.Google Scholar
  4. 4.
    Salles G, Barrett M, Foa R, Maurer J, O’Brien S, Valente N, et al. Rituximab in B-cell hematologic malignancies: a review of 20 years of clinical experience. Adv Ther. 2017;34(10):2232–73.CrossRefGoogle Scholar
  5. 5.
    Gurcan HM, Keskin DB, Stern JN, Nitzberg MA, Shekhani H, Ahmed AR. A review of the current use of rituximab in autoimmune diseases. Int Immunopharmacol. 2009;9(1):10–25.CrossRefGoogle Scholar
  6. 6.
    Sanz I. Indications of rituximab in autoimmune diseases. Drug Discov Today Ther Strateg. 2009;6(1):13–9.CrossRefGoogle Scholar
  7. 7.
    European Medicines Agency. Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: non-clinical and clinical issues. 2014. Accessed 20 Sep 2018.
  8. 8.
    World Health Organization. Guidelines on evaluation of monoclonal antibodies as similar biotherapeutic products (SBPs). 2016. Expert Committee on Biological Standardization. Accessed 18 Sep 2018.
  9. 9.
    US Food and Drug Administration. Guidance for industry: quality considerations in demonstrating biosimilarity of a therapeutic protein product to a reference product. 2015. Accessed 18 Sep 2018.
  10. 10.
    ANMAT. Disposición No. 7729/11. 2011. Accessed 10 Dec 2018.
  11. 11.
    Garcia R, Araujo DV. The regulation of biosimilars in Latin America. Curr Rheumatol Rep. 2016;18(3):16.CrossRefGoogle Scholar
  12. 12.
    Ministry of Public Health of Uruguay. Decreto No. 38/015. Documento de Registro de Medicamentos Biotecnológicos. 2015. Accessed 18 Dec 2018.Google Scholar
  13. 13.
    GaBI Online. Similar biotherapeutic products approved and marketed in Latin America. 2013. Accessed 14 Sep 2018.
  14. 14.
    National Institute of Public Health of Mexico. Norma Oficial Mexicana NOM-257-SSA1-2014, En materia de medicamentos biotecnológicos. 2014. Accessed 18 Dec 2018.
  15. 15.
    Anvisa. Resolução da Diretoria Colegiada_RDC No. 55. 2010.; Accessed 18 Dec 2018.
  16. 16.
    Butler M. Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals. Appl Microbiol Biotechnol. 2005;68(3):283–91.CrossRefGoogle Scholar
  17. 17.
    Hossler P, Khattak SF, Li ZJ. Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology. 2009;19(9):936–49.CrossRefGoogle Scholar
  18. 18.
    Cuello HA, Segatori VI, Alberto M, Pesce A, Alonso DF, Gabri MR. Comparability of antibody-mediated cell killing activity between a proposed biosimilar RTXM83 and the originator rituximab. BioDrugs Clin Immunother Biopharm Gene Ther. 2016;30(3):225–31.Google Scholar
  19. 19.
    Cartron G, Dacheux L, Salles G, Solal-Celigny P, Bardos P, Colombat P, et al. Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene. Blood. 2002;99(3):754–8.CrossRefGoogle Scholar
  20. 20.
    Hatjiharissi E, Xu L, Santos DD, Hunter ZR, Ciccarelli BT, Verselis S, et al. Increased natural killer cell expression of CD16, augmented binding and ADCC activity to rituximab among individuals expressing the Fc{gamma}RIIIa-158 V/V and V/F polymorphism. Blood. 2007;110(7):2561–4.CrossRefGoogle Scholar
  21. 21.
    Treon SP, Hansen M, Branagan AR, Verselis S, Emmanouilides C, Kimby E, et al. Polymorphisms in FcgammaRIIIA (CD16) receptor expression are associated with clinical response to rituximab in Waldenstrom’s macroglobulinemia. J Clin Oncol. 2005;23(3):474–81.CrossRefGoogle Scholar
  22. 22.
    Candelaria M, Gonzalez D, Fernandez Gomez FJ, Paravisini A, Del Campo Garcia A, Perez L, et al. Comparative assessment of pharmacokinetics, and pharmacodynamics between RTXM83, a rituximab biosimilar, and rituximab in diffuse large B-cell lymphoma patients: a population PK model approach. Cancer Chemother Pharmacol. 2018;81(3):515–27.CrossRefGoogle Scholar
  23. 23.
    Idusogie EE, Presta LG, Gazzano-Santoro H, Totpal K, Wong PY, Ultsch M, et al. Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc. J Immunol. 2000;164(8):4178–84.CrossRefGoogle Scholar
  24. 24.
    Lefranc M-P. IMGT®, the international ImMunoGeneTics information system®. Montpellier, France. Accessed 26 Mar 2019.
  25. 25.
    Beck A, Diemer H, Ayoub D, Debaene F, Wagner-Rousset E, Carapito C, et al. Analytical characterization of biosimilar antibodies and Fc-fusion proteins. TrAC Trends Anal Chem. 2013;48:81–95.CrossRefGoogle Scholar
  26. 26.
    Du Y, Walsh A, Ehrick R, Xu W, May K, Liu H. Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. mAbs. 2012;4(5):578–85.CrossRefGoogle Scholar
  27. 27.
    Liu H, Gaza-Bulseco G, Faldu D, Chumsae C, Sun J. Heterogeneity of monoclonal antibodies. J Pharm Sci. 2008;97(7):2426–47.CrossRefGoogle Scholar
  28. 28.
    Walsh G, Jefferis R. Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol. 2006;24(10):1241–52.CrossRefGoogle Scholar
  29. 29.
    Sinha S, Pipes G, Topp EM, Bondarenko PV, Treuheit MJ, Gadgil HS. Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs. J Am Soc Mass Spectrom. 2008;19(11):1643–54.CrossRefGoogle Scholar
  30. 30.
    Rustandi RR, Washabaugh MW, Wang Y. Applications of CE SDS gel in development of biopharmaceutical antibody-based products. Electrophoresis. 2008;29(17):3612–20.CrossRefGoogle Scholar
  31. 31.
    Clodfelter DK, Nussbaum MA, Reilly J. Comparison of free solution capillary electrophoresis and size exclusion chromatography for quantitating non-covalent aggregation of an acylated peptide. J Pharm Biomed Anal. 1999;19(5):763–75.CrossRefGoogle Scholar
  32. 32.
    den Engelsman J, Garidel P, Smulders R, Koll H, Smith B, Bassarab S, et al. Strategies for the assessment of protein aggregates in pharmaceutical biotech product development. Pharm Res. 2011;28(4):920–33.CrossRefGoogle Scholar
  33. 33.
    Gabrielson JP, Brader ML, Pekar AH, Mathis KB, Winter G, Carpenter JF, et al. Quantitation of aggregate levels in a recombinant humanized monoclonal antibody formulation by size-exclusion chromatography, asymmetrical flow field flow fractionation, and sedimentation velocity. J Pharm Sci. 2007;96(2):268–79.CrossRefGoogle Scholar
  34. 34.
    Grinberg N, Blanco R, Yarmush DM, Karger BL. Protein aggregation in high-performance liquid chromatography: hydrophobic interaction chromatography of beta-lactoglobulin A. Anal Chem. 1989;61(6):514–20.CrossRefGoogle Scholar
  35. 35.
    Montacir O, Montacir H, Eravci M, Springer A, Hinderlich S, Saadati A, et al. Comparability study of rituximab originator and follow-on biopharmaceutical. J Pharm Biomed Anal. 2017;5(140):239–51.CrossRefGoogle Scholar
  36. 36.
    Nupur N, Chhabra N, Dash R, Rathore AS. Assessment of structural and functional similarity of biosimilar products: rituximab as a case study. mAbs. 2018;10(1):143–58.CrossRefGoogle Scholar
  37. 37.
    Saxena A, Wu D. Advances in therapeutic Fc engineering—modulation of IgG-associated effector functions and serum half-life. Front Immunol. 2016;7:580.CrossRefGoogle Scholar
  38. 38.
    Visser J, Feuerstein I, Stangler T, Schmiederer T, Fritsch C, Schiestl M. Physicochemical and functional comparability between the proposed biosimilar rituximab GP2013 and originator rituximab. BioDrugs Clin Immunother Biopharm Gene Ther. 2013;27(5):495–507.Google Scholar
  39. 39.
    Harris RJ. Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture. J Chromatogr A. 1995;705(1):129–34.CrossRefGoogle Scholar
  40. 40.
    Antes B, Amon S, Rizzi A, Wiederkum S, Kainer M, Szolar O, et al. Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function. J Chromatogr B Anal Technol Biomed Life Sci. 2007;852(1–2):250–6.CrossRefGoogle Scholar
  41. 41.
    Dick LW Jr, Qiu D, Mahon D, Adamo M, Cheng KC. C-terminal lysine variants in fully human monoclonal antibodies: investigation of test methods and possible causes. Biotechnol Bioeng. 2008;100(6):1132–43.CrossRefGoogle Scholar
  42. 42.
    Hong J, Lee Y, Lee C, Eo S, Kim S, Lee N, et al. Physicochemical and biological characterization of SB2, a biosimilar of Remicade (R) (infliximab). mAbs. 2017;9(2):364–82.CrossRefGoogle Scholar
  43. 43.
    Khawli LA, Goswami S, Hutchinson R, Kwong ZW, Yang J, Wang X, et al. Charge variants in IgG1: isolation, characterization, in vitro binding properties and pharmacokinetics in rats. mAbs. 2010;2(6):613–24.CrossRefGoogle Scholar
  44. 44.
    European Medicines Agency. Guideline on similar biological medicinal products containing monoclonal antibodies—non-clinical and clinical issues. 2012. Accessed 10 Jan 2019.
  45. 45.
    Schiestl M, Stangler T, Torella C, Cepeljnik T, Toll H, Grau R. Acceptable changes in quality attributes of glycosylated biopharmaceuticals. Nat Biotechnol. 2011;29(4):310–2.CrossRefGoogle Scholar
  46. 46.
    Ghaderi D, Taylor RE, Padler-Karavani V, Diaz S, Varki A. Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nat Biotechnol. 2010;28(8):863–7.CrossRefGoogle Scholar
  47. 47.
    Varki A. Loss of N-glycolylneuraminic acid in humans: mechanisms, consequences, and implications for hominid evolution. Am J Phys Anthropol. 2001;33:54–69.CrossRefGoogle Scholar
  48. 48.
    Cerny T, Borisch B, Introna M, Johnson P, Rose AL. Mechanism of action of rituximab. Anticancer Drugs. 2002;13(Suppl 2):S3–10.CrossRefGoogle Scholar
  49. 49.
    Smith MR. Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance. Oncogene. 2003;22(47):7359–68.CrossRefGoogle Scholar
  50. 50.
    Weiner GJ. Rituximab: mechanism of action. Semin Hematol. 2010;47(2):115–23.CrossRefGoogle Scholar
  51. 51.
    Milone G, Penna M, Fernández F, Spitzer E, Millan S, De Caso P, et al. Post-marketing surveillance with a biosimilar of rituximab (Novex®) in Argentina. 59th ASH annual meeting; Atlanta; 2017. p. 2131.Google Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • María L. Cerutti
    • 1
    • 3
  • Analía Pesce
    • 1
  • Cédric Bès
    • 2
  • Mauricio Seigelchifer
    • 1
    Email author
  1. 1.mAbxienceBuenos AiresArgentina
  2. 2.mAbxienceMadridSpain
  3. 3.Fundación Instituto Leloir-IIBBA-CONICETBuenos AiresArgentina

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