Design of a strong cation exchange methodology for the evaluation of charge heterogeneity in glatiramer acetate

Campos-García, Víctor R.; López‐Morales, Carlos A.; Benites-Zaragoza, Eleuterio; Jiménez-Miranda, Armando; Garza, Carlos E. Espinosa-de la; Herrera-Fernández, Daniel; Padilla-Calderón, Jesús; Pérez, Nestor O.; Flores-Ortiz, Luis F.; Medina‐Rivero, Emilio

doi:10.1016/j.jpba.2016.10.002

Cited by 6 publications

(9 citation statements)

References 19 publications

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“…For the latter, multiple conditions in each manufacturing step, as well as purification stages, are tightly controlled to ensure consistent physicochemical composition. Even minimal modifications in conditions (e.g., time or the order of addition of the amino acids or temperature of the reaction) can alter composition . The results we describe above, from high‐resolution gene expression and other testing, seem to be at odds with some of the statements Anderson et al .…”

Section: Discussionmentioning

confidence: 73%

Compositional differences between Copaxone and Glatopa are reflected in altered immunomodulation ex vivo in a mouse model

Grossman

Kolitz²,

Komlosh

et al. 2017

Annals of the New York Academy of Sciences

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Copaxone (glatiramer acetate, GA), a structurally and compositionally complex polypeptide nonbiological drug, is an effective treatment for multiple sclerosis, with a well-established favorable safety profile. The short antigenic polypeptide sequences comprising therapeutically active epitopes in GA cannot be deciphered with state-of-the-art methods; and GA has no measurable pharmacokinetic profile and no validated pharmacodynamic markers. The study reported herein describes the use of orthogonal standard and high-resolution physicochemical and biological tests to characterize GA and a U.S. Food and Drug Administration-approved generic version of GA, Glatopa (USA-FoGA). While similarities were observed with low-resolution or destructive tests, differences between GA and USA-FoGA were measured with high-resolution methods applied to an intact mixture, including variations in surface charge and a unique, high-molecular-weight, hydrophobic polypeptide population observed only in some USA-FoGA lots. Consistent with published reports that modifications in physicochemical attributes alter immune-related processes, genome-wide expression profiles of ex vivo activated splenocytes from mice immunized with either GA or USA-FoGA showed that 7-11% of modulated genes were differentially expressed and enriched for immune-related pathways. Thus, differences between USA-FoGA and GA may include variations in antigenic epitopes that differentially activate immune responses. We propose that the assays reported herein should be considered during the regulatory assessment process for nonbiological complex drugs such as GA.

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Section: Discussionmentioning

confidence: 73%

Compositional differences between Copaxone and Glatopa are reflected in altered immunomodulation ex vivo in a mouse model

Grossman

Kolitz²,

Komlosh

et al. 2017

Annals of the New York Academy of Sciences

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“…Besides, GA intrinsic heterogeneity is utterly responsible for its immunological, pharmacological and toxicological profiles; thus confirming the relationship between its identity and biological activity. In this regard, is mandatory to use suitable techniques to differentiate among closely related profiles against the distinctive heterogeneity profile of GA, without missing its minor populations 46 . We developed SE-UPLC 45 , SCX-UPLC and RP-UPLC methodologies 46 to evaluate molecular mass, electric charge and hydrophobicity distributions as the most reliable characteristics of the whole mixture of amino acid sequences and peptide lengths of GA through each stage of the manufacturing process (viz., polymerization, depolymerization, deprotection and purification) in order to assess critical process parameters and their impact on GA structural signatures.…”

Section: Resultsmentioning

confidence: 99%

“…All experiments were modified from the standard synthesis conditions (STD) used for GA manufacturing. GA produced by STD (GA-STD, Probioglat) has been extensively characterized in terms of its distinctive and equivalent molecular mass 45 ; electric charge and hydrophobicity 46 ; and refractive index increment and extinction coefficient 51 with respect to the reference medicinal product (Copaxone).…”

Section: Resultsmentioning

confidence: 99%

“…GA complexity may impede the complete identification of the physicochemical and biological properties utterly responsible of its efficacy and safety 35 – 43 , but it does not preclude a finding of distinctive attributes that define GA towards dissecting its identity and improve the understanding of the biological and clinical properties of glatiramer acetate 44 . For instance, GA highly disperse molecular mass distribution has been characterized, ranging from ~3 kDa to ~45 kDa with a mass-average molar mass mean (M w ) of ~10.5 kDa 45 , along its highly disperse electric charge distribution that ranges from ~1 to more than 60(e) with a mean net positive charge of ~48(e) 46 .…”

Section: Introductionmentioning

confidence: 99%

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Process signatures in glatiramer acetate synthesis: structural and functional relationships

Campos-García¹,

Herrera-Fernández²,

Garza³

et al. 2017

Sci Rep

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Glatiramer Acetate (GA) is an immunomodulatory medicine approved for the treatment of multiple sclerosis, whose mechanisms of action are yet to be fully elucidated. GA is comprised of a complex mixture of polypeptides with different amino acid sequences and structures. The lack of sensible information about physicochemical characteristics of GA has contributed to its comprehensiveness complexity. Consequently, an unambiguous determination of distinctive attributes that define GA is of highest relevance towards dissecting its identity. Herein we conducted a study of characteristic GA heterogeneities throughout its manufacturing process (process signatures), revealing a strong impact of critical process parameters (CPPs) on the reactivity of amino acid precursors; reaction initiation and polymerization velocities; and peptide solubility, susceptibility to hydrolysis, and size-exclusion properties. Further, distinctive GA heterogeneities were correlated to defined immunological and toxicological profiles, revealing that GA possesses a unique repertoire of active constituents (epitopes) responsible of its immunological responses, whose modification lead to altered profiles. This novel approach established CPPs influence on intact GA peptide mixture, whose physicochemical identity cannot longer rely on reduced properties (based on complete or partial GA degradation), providing advanced knowledge on GA structural and functional relationships to ensure a consistent manufacturing of safe and effective products.

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“…A general strategy was developed to establish equivalence based on the overall scientific understanding of GA chemistry and synthesis process . Characterization of such complex drugs requires many physicochemical techniques to cover a wide range of structural information, including reversed‐phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), ion‐exchange chromatography, size‐exclusion chromatography, mass spectrometry (MS), capillary isoelectric focusing, and multiangle light scattering …”

Section: Introductionmentioning

confidence: 99%

Application of 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate derivatization for enhanced peptide mapping analysis of non‐biological complex drug glatiramer acetate using liquid chromatography/electrospray ionization collision‐induced dissociation high‐resolution mass spectrometry

Mesonzhnik

Kuznetsov

Bochkareva

et al. 2020

Rapid Comm Mass Spectrometry

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Rationale: Glatiramer acetate (GA) (Copaxone ® ) is a non-biological complex drug (NBCD) comprising random-sequence polymer chains of four amino acids (MW~5-9 kDa) with unknown structure. The characterization of NBCDs by reversed-phase liquid chromatography/mass spectrometry (RPLC/MS) peptide mapping is often impeded by insufficient separation and/or low sensitivity. To overcome this issue, pre-column derivatization of GA peptide digest was used to improve RPLC/MS detectability and to generate a comprehensive peptide profile.Methods: Amino groups of peptides generated by trypsin digestion of GA were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent.The derivatized mixture of random-sequence peptides was analyzed by liquid chromatography/positive-mode electrospray ionization collision-induced dissociation high-resolution mass spectrometry (RPLC/ESI-CID-HRMS/MS). Data-independent LC/MS E mode was used for data acquisition. Results:The derivatization of the GA peptide mixture increased the detectability of RPLC/ESI-CID-HRMS/MS analysis. The efficacy of the procedure was demonstrated by using selected peptides related to different polymeric chain origins. The resultant peptides were derivatized in a predictable manner giving a minimum of side products.The reproducibility of the developed method was demonstrated by comparing peptide elution profiles derived from six Copaxone ® lots. Conclusions:Application of the AQC pre-column derivatization provides a framework that could be used as an attractive approach for monitoring the quality and characterization of NBCD products in the pharmaceutical industry.

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Design of a strong cation exchange methodology for the evaluation of charge heterogeneity in glatiramer acetate

Cited by 6 publications

References 19 publications

Compositional differences between Copaxone and Glatopa are reflected in altered immunomodulation ex vivo in a mouse model

Compositional differences between Copaxone and Glatopa are reflected in altered immunomodulation ex vivo in a mouse model

Process signatures in glatiramer acetate synthesis: structural and functional relationships

Application of 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate derivatization for enhanced peptide mapping analysis of non‐biological complex drug glatiramer acetate using liquid chromatography/electrospray ionization collision‐induced dissociation high‐resolution mass spectrometry

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