Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping

Chloupek, Rosanne C.; Harris, Reed J.; Leonard, C K; Keck, Rodney G.; Keyt, B. A.; Spellman, Michael W.; Jones, Andrew J. S.; Hancock, William S.

doi:10.1016/s0021-9673(01)84491-5

Cited by 41 publications

(8 citation statements)

References 14 publications

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“…20,21 The most common technique is the enzymatic digestion of the reduced and alkylated antibodies with trypsin for the generation of peptides for LC/MS analysis. 22,23 Figure 2. the catumaxomab sample was analyzed using Rp-HpLC mass spectrometry using a pLRp-S column (Varian) with an acetonitrile gradient from 27% to 42% over 40 minutes on a 1200 HpLC system (Agilent) for separation, coupled with an Agilent 6220 eSI-toF mass spectrometer. the column temperature was 65°C and the flow rate was 200 µL/min.…”

Section: Resultsmentioning

confidence: 99%

Structural and functional characterization of the trifunctional antibody catumaxomab

Chelius¹,

Rosenberger²,

Gruber³

et al. 2010

mAbs

125

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Section: Resultsmentioning

confidence: 99%

Structural and functional characterization of the trifunctional antibody catumaxomab

Chelius¹,

Rosenberger²,

Gruber³

et al. 2010

mAbs

125

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“…For the digestion of low levels of IGF‐1, different proteolytic enzymes were evaluated. Trypsin is most often employed for protein digestions8 and a typical method was employed 9. The results however indicated a low digestion yield and non‐specific enzyme cleavage which could not be identified.…”

Section: Resultsmentioning

confidence: 99%

Growth hormone abuse in the horse: preliminary assessment of a mass spectrometric procedure for IGF‐1 identification and quantitation

Kock¹,

Rodgers²,

Swanepoel³

2001

Rapid Comm Mass Spectrometry

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Previous studies have shown that insulin-like growth factor 1 (IGF-1) is a promising marker for the detection of growth hormone (GH) abuse in the horse. The significant increases observed with GH administration in comparison to natural levels imply the possibility of setting a threshold level for IGF-1 that would be indicative of GH abuse. Although an immunoradiometric assay (IRMA) has been identified as a reliable screening method, a more specific IGF-1 quantification method needs to be developed for the prosecution of GH abuse by horseracing authorities. This study describes such an HPLC electrospray mass spectrometry (LC/ESI-MS) method that was developed and then assessed for the specific analysis of IGF-1 at the low levels encountered in serum. The structural identity of IGF-1 was confirmed by endoproteinase Asp-N digestion followed by LC/MS and LC/MS/MS characterisation. This was followed by quantification of IGF-1 as the intact molecule against an internal standard.

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“…Как правило, рекомбинантный tPA синтезируется в виде смеси молекул обоих типов в соотношении, близком к единице [6]. Функциональные различия между формами выражаются в аффинности к фибрину (II тип связывает лучше), а также на уровне скорости выведения препарата из организма [7][8][9].…”

Section: результаты и обсуждениеunclassified

Physico-Chemical and Biological Properties of Biosimilar and Reference Tissue Plasminogen Activator Products

Gusarova¹,

Пантюшенко²,

Simonov³

et al. 2019

Biopreparaty. Profilaktika, diagnostika, lechenie

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Recombinant tissue plasminogen activator (international nonproprietary name — alteplase) which was developed by «GENERIUM» (Russia) and received a marketing authorisation in Russia is completely analogous to Actilyse® which is used to treat medical conditions accompanied by thrombosis, such as acute myocardial infarction, pulmonary embolism, and ischemic stroke. The aim of the study was to carry out a comprehensive comparison of physico-chemical and biological properties of Revelyse® and the reference product Actilyse® in order to assess their biosimilarity. Materials and Methods: comparative peptide mapping and determination of comparability of chromatographic profiles of tryptic hydrolysates was performed using RP-HPLC and massspectrometry; the molecular weight distribution was determined by mass-spectrometry and polyacrylamide gel electrophoresis (Laemmli method). The purity and homogeneity of products as well as the content of related impurities (oligomers and fragments) were determined using gel filtration; N-glycosylation profile was analysed by hydrophilic HPLC, total sialic acid was quantified by the Svennerholm resorcinol method. Protein binding to fibrin and human fibrinogen was assessed by surface plasmon resonance, and the specific activity was compared by fibrin clot lysis. Results: the research demonstrated a complete overlap of the products’ peptide maps, which indicates the identity of аlteplase amino acid sequences in the two medicines being compared. The authors of the study also determined the molecular weight and the content of the intact single-stranded form of the protein, and quantified post-translational modifications, the content of sialic acids and neutral sugars. The analysis of the N-glycosylation profile revealed insignificant differences in the percentage of multiantenna complex glycans. The specificity of alteplase was evaluated by analysing the formation of protein complexes with natural alteplase ligands – fibrin and plasminogen activator inhibitor-1, but no significant differences were found. The comparison of specific activation of plasminogen fibrinolytic activity was performed based on the results of the assay analysing the fibrin clot lysis rate, and it demonstrated comparability of Revelyse® and Actilyse®. Conclusions: comparative experimental studies have shown no differences in the structure, charge distribution heterogeneity, impurities content, and specific activity of alteplase as a component of Revelyse® and the reference product Actilyse®, which leads to the conclusion that they are similar in terms of physicochemical and biological properties.

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Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping

Cited by 41 publications

References 14 publications

Structural and functional characterization of the trifunctional antibody catumaxomab

Structural and functional characterization of the trifunctional antibody catumaxomab

Growth hormone abuse in the horse: preliminary assessment of a mass spectrometric procedure for IGF‐1 identification and quantitation

Physico-Chemical and Biological Properties of Biosimilar and Reference Tissue Plasminogen Activator Products

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