Conformational Comparability of Factor IX–Fc Fusion Protein, Factor IX, and Purified Fc Fragment in the Absence and Presence of Calcium

Houde, Damian J.; Berkowitz, Steven A.

doi:10.1002/jps.23064

Cited by 25 publications

(16 citation statements)

References 48 publications

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“…Additionally, many novel recombinant proteins are being developed and released to the pharmaceutical market as therapeutic drugs. These recombinant therapeutics contain multiple modifications such as missense mutations, synonymous mutations, and deletions [3,4]. …”

Section: Introductionmentioning

confidence: 99%

A Gene-Specific Method for Predicting Hemophilia-Causing Point Mutations

Hamasaki-Katagiri

Salari

et al. 2013

Journal of Molecular Biology

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A fundamental goal of medical genetics is the accurate prediction of genotype–phenotype correlations. As an approach to develop more accurate in silico tools for prediction of disease-causing mutations of structural proteins, we present a gene- and disease-specific prediction tool based on a large systematic analysis of missense mutations from hemophilia A (HA) patients. Our HA-specific prediction tool, HApredictor, showed disease prediction accuracy comparable to other publicly available prediction software. In contrast to those methods, its performance is not limited to non-synonymous mutations. Given the role of synonymous mutations in disease and drug codon optimization, we propose that utilizing a gene- and disease-specific method can be highly useful to make functional predictions possible even for synonymous mutations. Incorporating computational metrics at both nucleotide and amino acid levels along with multiple protein sequence/structure alignment significantly improved the predictive performance of our tool. HApredictor is freely available for download at http://www.ncbi.nlm.nih.gov/CBBresearch/Przytycka/HA_Predict/index.htm.

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Section: Introductionmentioning

confidence: 99%

A Gene-Specific Method for Predicting Hemophilia-Causing Point Mutations

Hamasaki-Katagiri

Salari

et al. 2013

Journal of Molecular Biology

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“…28 This hypothesis is in accordance with the propensity of Fc fusion proteins to form protein aggregates, 28 which is mostly induced by hydrophobic interactions. 29 Indeed, it has been shown that the coupling of the Fc region to the hFIX resulted in a difference in hydrogen atom exposition of residues 408-414, 30 which potentially favoured hydrophobic interactions and subsequent formation of hFIX-Fc fusion protein aggregates.…”

Section: Covalent Fusion Of the Fc Domain Of Human Immunoglobulin Igg1mentioning

confidence: 99%

Potential limits of AAV‐based gene therapy with the use of new transgenes expressing factor IX fusion proteins

et al. 2018

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Introduction The variety of treatment for haemophilia B (HB) has recently improved with the emergence of both AAV‐based gene therapy and bioengineered human factor IX (hFIX) molecules with prolonged half‐life due to fusion to either albumin (Alb) or immunoglobulin Fc fragment (Fc). Aim Adeno‐associated viral vectors (AAV) mediating expression of hFIX‐Alb and hFIX‐Fc fusion proteins was investigated for gene therapy of HB to explore if their extended half‐life translates to higher plasma levels of FIX. Methods Single‐stranded cross‐packaged AAV2/8 vectors expressing hFIX‐Alb, hFIX‐Fc and hFIX were evaluated in vitro, and in mice. Results Both hFIX‐Alb and hFIX‐Fc fusion proteins were synthesized and expressed as single chains of expected size following AAV‐mediated gene transfer in vitro and in vivo. The procoagulant properties of these hFIX‐fusion proteins were comparable to wild‐type hFIX. However, their expression levels were threefold lower than wild‐type hFIX in vivo most likely due to inefficient secretion. Conclusion This, the first, evaluation of hFIX‐fusion proteins in the context of AAV gene transfer suggests that the hFIX‐fusion proteins are secreted inefficiently from the liver, thus preventing their optimal use in gene therapy approaches.

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“…In some cases, due to the complexity and limitations of NMR and X-ray crystallography techniques, routine biopharmaceutical analyses of conformation and stability of protein therapeutics typically rely only on classical biophysical methods, such as Circular Dichroism, FTIR spectroscopy, calorimetry and size exclusion chromatography which are less time consuming but only provide limited, global structural information. HDX-MS has emerged as a more informative method for higher order structure characterization, and it is especially suitable for comparability studies detecting more subtle structural differences and offering localized information about protein segments with unexpected protection levels [56–59]. This method is used as an orthogonal measurement along with other biophysical techniques in structural comparability studies for drug development.…”

Section: Comparability Studiesmentioning

confidence: 99%

Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications

Wei

Tao

et al. 2014

Drug Discovery Today

183

157

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The higher order structure of protein therapeutics can be interrogated with hydrogen/deuterium exchange mass spectrometry (HDX-MS). HDX-MS is now a widely used tool in the structural characterization of protein therapeutics. In this article, HDX-MS based workflows designed for both protein therapeutic discovery and development processes are presented, focusing on the specific applications of epitope mapping for protein/drug interactions and biopharmaceutical comparability studies. Future trends in the application of HDX-MS to protein therapeutics characterization are also described.

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Conformational Comparability of Factor IX–Fc Fusion Protein, Factor IX, and Purified Fc Fragment in the Absence and Presence of Calcium

Cited by 25 publications

References 48 publications

A Gene-Specific Method for Predicting Hemophilia-Causing Point Mutations

A Gene-Specific Method for Predicting Hemophilia-Causing Point Mutations

Potential limits of AAV‐based gene therapy with the use of new transgenes expressing factor IX fusion proteins

Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications

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