Determination of pegfilgrastim aggregates by size-exclusion high-performance liquid chromatography on a methacrylate-based column

Shahbazi, Majid; Zahedy, Elnaz Tamaskany; Kiumarsi, Shiva; Soltanabad, Mojtaba Hadi; Azar, Saleh Shahbazi; Amini, Hossein

doi:10.1016/j.biologicals.2017.02.003

Cited by 3 publications

(4 citation statements)

References 13 publications

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“…An increase in impurity content at RRT about 0.83 and RRT about 0.87 was observed after vortexing (S23 Fig and Table J in S1 Appendix). The SEC-MALS data of RRT about 0.87 species in the vortexed samples of pegfilgrastim had a molecular weight of 91.4 kDa while the main peak molecular weight is 45.6 kDa [22, 23]. This indicates that the RRT ~ 0.87 impurity is a dimer of pegfilgrastim.…”

Section: Resultsmentioning

confidence: 99%

Structural similarity, characterization of Poly Ethylene Glycol linkage and identification of product related variants in biosimilar pegfilgrastim

et al. 2019

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The approval of biosimilars requires demonstration of biosimilarity, which rests on the base of thorough analytical characterization of the biosimilar product. In addition to demonstration of biosimilarity, the product related impurities need to be thoroughly characterized and controlled at minimal levels. Pegylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG (Poly Ethylene Glycol) heterogeneity, site of addition and number of attached pegylated moieties. A combination of several methods including circular dichroism, FTIR (Fourier-transform Infrared Spectroscopy), fluorescence spectroscopy, DSC (Differential Scanning Calorimetry), 1D and 2D NMR (Nuclear Magnetic Resonance), Edman degradation and peptide mapping by LC-MS (Liquid Chromatography Mass Spectrometry) were used for characterization of N-terminally pegylated filgrastim. Product related impurities such as oxidized, reduced, deamidated, dipegylated variants and monopegylated positional isomers have been characterized in detail using various HPLC (High Performance Liquid Chromatography) based methods and LC-MS techniques. The functional characterization in terms of receptor binding and cell proliferation assay was conducted for the similarity assessment and the potential impact of the product variants on the in vitro biological activity has also been assessed. In summary, this study presents, for the first time, a detailed structural and molecular level characterization of a biosimilar pegfilgrastim providing a strong base for the demonstration of overall biosimilarity of the product with the innovator product.

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Section: Resultsmentioning

confidence: 99%

Structural similarity, characterization of Poly Ethylene Glycol linkage and identification of product related variants in biosimilar pegfilgrastim

et al. 2019

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“…Aggregation is considered as the most common physical instability of proteins occurring during manufacture and storage. Several strategies have transpired to augment the virtues of protein [31]. The most successful strategy is PEGylation, which refers to the conjugation of one or more PEG molecules to macromolecules.…”

Section: Discussionmentioning

confidence: 99%

Site-Specific PEGylation of Recombinant Immunotoxin DAB389IL-2: Structural and Functional Assessment

et al. 2019

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Background: DAB 389 IL-2 is considered a fusion immunotoxin and used for the treatment of Cutaneous T Cell Lymphoma (CTCL). It is composed of two distinct portions; the catalytic domain of diphtheria toxin and IL-2. Because of DAB 389 IL-2 free cysteine residue (Cys 513 in IL-2 part), it is prone to unwanted intramolecular and intermolecular disulfide bonds formation and aggregation problems. Aggregation is considered as the most common physical instability. PEGylation is a practical approach to increase the stability and half-life of therapeutic proteins. Materials and Methods: In this study, the PEGylation of recombinant DAB 389 IL-2 was performed by mPEG-vinylsulfone, through partial denaturation condition at 4° C for 24 h. To confirm the PEGylation reaction, SDS-PAGE and Dynamic Light Scattering (DLS) was used. The structure of DAB 389 IL-2 and its PEGylated immunotoxin form were analyzed using the Circular Dichroism (CD) and fluorescence methods. Also, the K562 cells line were treated with various concentrations of DAB 389 IL-2 and conjugated form. In the following, the nuclease activities of DAB 389 IL-2 and PEGylated form were determined. Results: The SDS-PAGE result confirmed the site-specific PEGylation of DAB 389 IL-2. Spectroscopy results exhibited that the PEGylation does not affect the protein original structure. Also, the cytotoxicity assay and nuclease activity test confirmed that PEGylated protein induces death in K562 cells line and DNA degradation, respectively. Conclusion: PEGylated immunotoxin DAB 389 IL-2 has a proper structure and function; thus, PEGylated immunotoxin requires more study because of its unique properties.

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“…One of the most challenging problems in pharmaceutical monoclonal antibody formulations is protein aggregation, which is directly associated with the quality of the final product, wherein high-molecularweight aggregates form and can induce immunogenicity (Hernández-Jiménez et al, 2018). Size exclusion liquid chromatography (SEC) is commonly used and acknowledged as the best analytical methodology for the quantitation of the native protein and to monitor potential aggregation because it allows for characterization with minimal impact on the conformational structure (Stamm et al, 2013;Shahbazi et al, 2017;Perobelli et al, 2018).…”

Section: Quantitation Of Certolizumab Pegol By Validated Liquid Chrom...mentioning

confidence: 99%

“…Other additives may also be needed, and it is well known that polar organic compounds, such as ethanol, can reduce protein packaging interactions (Ratto et al, 1997;Goyon et al, 2017;Shahbazi et al, 2017).…”

Section: Optimization Of Chromatographic Conditionsmentioning

confidence: 99%

Quantitation of certolizumab pegol by validated liquid chromatography methods

Cardoso Júnior,

Xavier,

Dumoncel

et al. 2023

Braz. J. Pharm. Sci.

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Certolizumab pegol (CZP) is a Fab' fragment of the humanized antibody with anti-TNF-α activity that is indicated as therapy for Crohn's disease and rheumatoid arthritis. Using a BioSep-SEC-S3000 column (300 x 4.6 mm i.d., 5 µm particle size), a size exclusion liquid chromatography (SEC) method was developed. Mobile phase A consisted of 100 mM monobasic sodium phosphate and 200 mM sodium chloride (pH 7.0), while mobile phase B was ethanol (95:5, v/v), and the analysis was performed using a diode array detector (DAD) set to 214 nm and a flow rate of 0.5 ml min -1 . In addition, a reversed-phase liquid chromatography (RP-LC) method based on gradient elution was developed on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d., 3.5 µm particle size) kept at 80 °C. Mobile phase A was 0.1% (v/v) TFA in ultrapure water, and mobile phase B was a mixture of propanol, acetonitrile, ultrapure water and TFA (70 + 20 + 9.9 + 0.1, v/v) operated at a flow rate of 1.0 ml min -1 , and DAD was applied at 214 nm. CZP elution was achieved with retention times of 5.6 min and 9.0 min for SEC and RP-LC, respectively.

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Determination of pegfilgrastim aggregates by size-exclusion high-performance liquid chromatography on a methacrylate-based column

Cited by 3 publications

References 13 publications

Structural similarity, characterization of Poly Ethylene Glycol linkage and identification of product related variants in biosimilar pegfilgrastim

Structural similarity, characterization of Poly Ethylene Glycol linkage and identification of product related variants in biosimilar pegfilgrastim

Site-Specific PEGylation of Recombinant Immunotoxin DAB389IL-2: Structural and Functional Assessment

Quantitation of certolizumab pegol by validated liquid chromatography methods

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