Studying the formation of aggregates in recombinant human granulocyte-colony stimulating factor (rHuG-CSF), lenograstim, using size-exclusion chromatography and SDS-PAGE

Ribarska, Jasmina Tonic; Jolevska, Suzana Trajkovic; Panovska, Ana Poceva; Dimitrovska, Aneta

doi:10.2478/v10007-008-0003-6

Cited by 9 publications

(12 citation statements)

References 17 publications

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“…SDS-PAGE is a well-established technique to investigate whether the aggregates formed were composed of covalently and/or non-covalently linked species (4,38). …”

Section: Resultsmentioning

confidence: 99%

Mass Spectrometric Analysis of Intact Human Monoclonal Antibody Aggregates Fractionated by Size-Exclusion Chromatography

et al. 2010

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PurposeThe aim of this study was to develop a method to characterize intact soluble monoclonal IgG1 antibody (IgG) oligomers by mass spectrometry.MethodsIgG aggregates (dimers, trimers, tetramers and high-molecular-weight oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatography (HP-SEC), dialyzed against ammonium acetate pH 6.0 (a mass spectrometry-compatible volatile buffer), and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS).ResultsMonomeric and aggregated IgG fractions in the stressed IgG formulation were successfully isolated by HP-SEC. ESI-TOF MS analysis enabled us to determine the molecular weight of the monomeric IgG as well as the aggregates, including dimers, trimers and tetramers. HP-SEC separation and sample preparation proved to be necessary for good quality signal in ESI-TOF MS. Both the HP-SEC protocol and the ESI-TOF mass spectrometric technique were shown to leave the IgG oligomers largely intact.ConclusionsESI-TOF MS is a useful tool complementary to HP-SEC to identify and characterize small oligomeric protein aggregates.

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“…SDS-PAGE is a well-established technique to investigate whether the aggregates formed were composed of covalently and/or non-covalently linked species (4,38). …”

Section: Resultsmentioning

confidence: 99%

Mass Spectrometric Analysis of Intact Human Monoclonal Antibody Aggregates Fractionated by Size-Exclusion Chromatography

et al. 2010

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“…Agitation and successive freeze-thaw cycles were also assessed under both accelerated and quiescent long-term storage conditions [11,12,[17][18][19]. On the other hand, very few studies covering the analysis of LGM are reported such as RP-HPLC [20] and SE-HPLC [21,22]. However, these studies lack proper method validation and are either not stability-indicating or do not provide in-depth evaluation of the stability of LGM using orthogonal testing protocols.…”

Section: Introductionmentioning

confidence: 99%

Assessment of the Effects of Glycosylation on the Pattern and Kinetics of Degradation of Lenograstim in Comparison to Filgrastim Using a Stability-Indicating Orthogonal Testing Protocol

et al. 2015

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“…Therefore, we were inspired to develop a ligation method that would provide G-CSF 74–174 directly, without postligative manipulation of an aggregation-prone sequence. 13 …”

Section: Introductionmentioning

confidence: 99%

“…The structural complexity, size, and physical properties 13 of aglycone 2a present a synthetic challenge addressed by the advance of a solution-phase ligation of large, side-chain-unprotected polypeptides (>20-mer residue sequences). 27 This effort builds upon previous research in our laboratory pertaining to the intriguing reactivity of isonitriles with carboxylic and thiocarboxylic acids alike.…”

Section: Introductionmentioning

confidence: 99%

Fully Synthetic Granulocyte Colony-Stimulating Factor Enabled by Isonitrile-Mediated Coupling of Large, Side-Chain-Unprotected Peptides

Roberts¹,

Johnston²,

Shieh³

et al. 2015

J. Am. Chem. Soc.

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Human granulocyte colony-stimulating factor (G-CSF) is an endogenous glycoprotein involved in hematopoiesis. Natively glycosylated and nonglycosylated recombinant forms, lenograstim and filgrastim, respectively, are used clinically to manage neutropenia in patients undergoing chemotherapeutic treatment. Despite their comparable therapeutic potential, the purpose of O-linked glycosylation at Thr133 remains a subject of controversy. In light of this, we have developed a synthetic platform to prepare G-CSF aglycone with the goal of enabling access to native and designed glycoforms with site-selectivity and glycan homogeneity. To address the synthesis of a relatively large, aggregation-prone sequence, we advanced an isonitrile-mediated ligation method. The chemoselective activation and coupling of C-terminal peptidyl Gly thioacids with the N-terminus of an unprotected peptide provide ligated peptides directly in a manner complementary to that with conventional native chemical ligation–desulfurization strategies. Herein, we describe the details and application of this method as it enabled the convergent total synthesis of G-CSF aglycone.

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Studying the formation of aggregates in recombinant human granulocyte-colony stimulating factor (rHuG-CSF), lenograstim, using size-exclusion chromatography and SDS-PAGE

Cited by 9 publications

References 17 publications

Mass Spectrometric Analysis of Intact Human Monoclonal Antibody Aggregates Fractionated by Size-Exclusion Chromatography

Mass Spectrometric Analysis of Intact Human Monoclonal Antibody Aggregates Fractionated by Size-Exclusion Chromatography

Assessment of the Effects of Glycosylation on the Pattern and Kinetics of Degradation of Lenograstim in Comparison to Filgrastim Using a Stability-Indicating Orthogonal Testing Protocol

Fully Synthetic Granulocyte Colony-Stimulating Factor Enabled by Isonitrile-Mediated Coupling of Large, Side-Chain-Unprotected Peptides

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