Selective functional activity measurement of a PEGylated protein with a modification-dependent activity assay

Weber, Alfred; Engelmaier, Andrea; Mohr, Gabriele; Haindl, Sonja; Schwarz, Hans; Turecek, Peter L.

doi:10.1016/j.jpba.2016.09.045

Cited by 4 publications

(3 citation statements)

References 26 publications

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“…in clinical monitoring) . Indeed, product‐specific standards are currently under evaluation for another PEGylated rFVIII (N8‐GP) and the selective measurement of functional BAX 855 has also recently been described .…”

Section: Resultsmentioning

confidence: 99%

Impact of a product‐specific reference standard for the measurement of a PEGylated rFVIII activity: the Swiss Multicentre Field Study

Bulla

Poncet

Alberio³

et al. 2017

Haemophilia

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Section: Resultsmentioning

confidence: 99%

Impact of a product‐specific reference standard for the measurement of a PEGylated rFVIII activity: the Swiss Multicentre Field Study

Bulla

Poncet

Alberio³

et al. 2017

Haemophilia

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“…Since activation and complex formation of FIX Padua was apparently not hampered considerably, there was no steric hindrance imposed by the binding of the antibody to an epitope of FIX Padua that included amino acid L338. Our approach of combining antibody capture with selective activity measurement of the mutant protein variant resembles that described for the measurement of PEGylated factor VIII, 31 using the polyethylene glycol (PEG) moiety for the selective capture of the modified protein. The sensitivity of the assay calibration curve (0.2 mU/mL) of the chromogenic FIX assay was 50 times higher than that described.…”

Section: Discussionmentioning

confidence: 99%

Development of Methods for the Selective Measurement of the Single Amino Acid Exchange Variant Coagulation Factor IX Padua

Weber¹,

Engelmaier²,

Voelkel³

et al. 2018

Molecular Therapy - Methods & Clinical Development

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The description of hyper-functional factor IX (FIX) Padua triggered the development of BAX 335, an AAV8-based hemophilia B gene therapy vector designed to compensate for low FIX protein expression levels by expressing the FIX Padua variant, thereby reducing the exposure to viral vector. The presence of inactive FIX protein at baseline hindered conventional FIX:Ag ELISA from contributing to a more profound understanding of clinical data from the BAX 335 Phase 1/2 study (ClinicalTrials.gov: NCT01687608). By applying phage display technology, a Fab2 mini-antibody selectively binding to FIX Padua was developed and used to establish a FIX Padua-specific ELISA. The assay adequately performed, utilizing human and monkey plasma samples, and enabled the selective quantification of FIX Padua protein in human plasma samples from the BAX 335 trial. The mini-antibody also allowed the development of a chromogenic FIX Padua-specific activity assay, which adequately performed in human and mouse plasma. Collectively, the isolated FIX Padua-specific mini-antibody enabled the development of transgene-product-specific assays, which should improve the monitoring of hemophilia B gene therapies. The approach applied here for FIX Padua could be leveraged to develop variant-specific activity assays for other therapies where highly active enzymes are instrumental in achieving therapeutic levels of the transgene product.

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“…19,20 Recently, a new type of chromogenic assay, termed the modification-dependent activity assay (MDAA), 21 has been developed. This assay enables selective measurement of PEGylated [22][23][24] or polysialylated [25][26][27] recombinant FVIII in the presence of nonmodified FVIII. The MDAA uses a modification-recognizing capturing agent, which currently is an antibody against polyethylene glycol or polysialic acid that selectively binds modified FVIII in the presence of endogenous, nonmodified FVIII.…”

Section: Introductionmentioning

confidence: 99%

Selective human factor VIII activity measurement after analytical in‐line purification

Engelmaier

Schrenk²,

Billwein³

et al. 2022

Research and Practice in Thrombosis and Haemostasis

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Background It is essential to measure the activity of factor VIII (FVIII) throughout the life cycle of a coagulation FVIII concentrate. Such measurement in nonclinical pharmacokinetic studies is potentially biased by the presence of endogenous nonhuman FVIII, and certain manufacturing process–related additives can also impact the assay performance. Finally, the presence of FVIII activity–mimicking antibodies poses challenges when measuring FVIII in samples. Therefore, we developed an antibody‐based chromogenic FVIII assay, which facilitates the selective and sensitive activity measurement of human FVIII in the presence of animal plasma and interfering agents. Methods Plate‐bound monoclonal anti‐FVIII antibody specifically captured human FVIII, which was then measured with a chromogenic activity assay. A human reference plasma preparation was used to construct the calibration curve. Spike recovery was carried out in a citrated cynomolgus monkey plasma–solvent/detergent mixture and in the presence of the bispecific antibody emicizumab. Results The calibration curve ranged from 3.03 to 97.0 mIU FVIII/ml and was obtained repeatedly with good accuracy. B domain–deleted and full‐length FVIII did not differ in their responses. Recovery of spiked human FVIII in citrated cynomolgus monkey plasma was 102.7%, while neither native monkey plasma nor the other animal specimen tested showed any activity. Solvent/detergent solution and the bispecific antibody emicizumab had no influence on the assay. Conclusion Combining antibody‐mediated specific capture of human FVIII and a chromogenic activity assay resulted in a selective and sensitive measurement of human FVIII with no interference by endogenous, nonhuman FVIII, manufacturing process additives, or an FVIII activity–mimicking antibody.

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Selective functional activity measurement of a PEGylated protein with a modification-dependent activity assay

Cited by 4 publications

References 26 publications

Impact of a product‐specific reference standard for the measurement of a PEGylated rFVIII activity: the Swiss Multicentre Field Study

Impact of a product‐specific reference standard for the measurement of a PEGylated rFVIII activity: the Swiss Multicentre Field Study

Development of Methods for the Selective Measurement of the Single Amino Acid Exchange Variant Coagulation Factor IX Padua

Selective human factor VIII activity measurement after analytical in‐line purification

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