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  • 1
    Online Resource
    Online Resource
    Biochemical and Functional Characterization of a Serum-Free rVWF Drug Candidate.
    American Society of Hematology ; 2006
    In:  Blood Vol. 108, No. 11 ( 2006-11-16), p. 1017-1017
    Details
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 1017-1017
    Abstract: Recombinant VWF was co-expressed with recombinant FVIII in CHO cells. In order to obtain fully processed, mature rVWF, rVWF was exposed to recombinant CHO-cell-derived furin for VWF pro-peptide removal. Fermentation for both rVWF and furin and downstream processing were performed under serum-free conditions. Mature rVWF was highly purified by a series of conventional chromatography steps to a purity of & gt; 95%. rVWF was formulated in a protein-free buffer. Several large scale batches of rVWF were produced and characterized in comparison to Humate-P, Wilfactin and a highly purified plasma-derived (pd)VWF, an experimental pd VWF product obtained from cryoprecipitate. Residual pro-VWF levels were approximately 2% of total VWF antigen compared with 0.12, 0.05 and 0.13 for the rVWF product, experimental pd VWF, Humate-P and Wilfactin, respectively. VWF pro-peptide levels were approximately 0.5 pmol per unit VWF antigen, compared with 0.6 and 0.9 for Humate-P and Wilfactin, respectively; in the experimental pd VWF pro-peptide was below the limit of detection. Residual FVIII content was approximately 0.02 units (chromogenic assay) per unit VWF antigen, compared with 0.4 units in Humate-P, and 0.04 units in Wilfactin, and ≤ 0.01 in the experimental pd VWF product. Ristocetin cofactor activity was around 0.7 VWF:RCo units per unit antigen for all rVWF and pd VWF products tested. Collagen-binding activity was also similar between rVWF and pd VWF controls (around 0.8 – 0.9 units per unit). FVIII-binding capacity of rVWF was compared by setting the mean pd VWF as 100% and was found to also be ± 100%. The VWF affinity for FVIII was similar in rVWF and pd VWF (Biacore). rVWF showed high molecular weight multimers in low resolution agarose gels, with a pre-dominance in the high molecular weight range. In contrast to pd VWF, rVWF showed homogenous multimer bands without triplet structure formation due to the non-exposure to ADAMTS13 during the manufacturing process. The pharmacokinetic properties of rVWF were compared with pd VWF in VWF knock-out mice. The half-life or rVWF in VWD mice was 4.3 and 3.5 hours for 2 batches investigated, which seems longer than the half-lives obtained for pd VWF, 2.9 hours for Humate-P and 2.2 hours for a highly purified pd VWF control. Both VWF preparations stabilized endogenous murine FVIII which rose over time to the levels observed in normal C57BI/6J mice. Overall, these results showed that the properties of mature, furin-processed rVWF in vitro and in vivo are equivalent to those of plasma-derived VWF.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V108.11.1017.1017
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 2
    Online Resource
    Online Resource
    PEG Modified rVWF Prolongs the Survival of Native rFVIII in Hemophilia A Knock-Out Mice.
    American Society of Hematology ; 2006
    In:  Blood Vol. 108, No. 11 ( 2006-11-16), p. 1002-1002
    Details
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 1002-1002
    Abstract: Mature CHO cell derived and highly purified rVWF was chemically derivatized by binding of 20 kDa polyethylene glycol (PEG) succinimidyl glutarate, to the primary amino groups in rVWF with covalent bond formation, primarily with lysine residues. PEG conjugates of rVWF were further purified and pharmaceutically formulated for application into hemophilia A knock-out mice. Groups of 6 mice each were treated with respective test and control articles to measure pharmacokinetics. Mice were treated with chemically modified rVWF in combination with recombinant human FVIII (rFVIII, Advate). For control purposes, mice were treated with unmodified rVWF plus rFVIII. Citrated plasma was prepared from their blood and measured for FVIII activity with a chromogenic FVIII assay. Mice received 300 IU/kg rFVIII together with 3.6 mg/kg PEG-rVWF or 1.6 mg/kg unmodified rVWF. FVIII levels were measured at time points 5 minutes and 3, 9, 24 and 32 hours after injection. FVIII levels found 5 minutes after injection were set as 100%. At all time points FVIII levels were substantially higher in the groups co-administered with PEG-rVWF. Mice co-administered with PEG-rVWF showed a FVIII level of 3% at 24 hours which was maintained at 2% after 32 hours, while in the controls treated with rFVIII and unmodified rVWF, FVIII levels were close to the limit of detection at 24 hours (~ 0,6%) and had completely disappeared at 32 hours. Assuming a one compartmental model, a mean half life of 4.4 hrs of rFVIII in the presence of PEG-rVWF and 1.2 hrs in the presence of unmodified rVWF was calculated (Scientist program, MicroMath, Saint Louis, MO, USA). The FVIII AUC was increased by a factor of 2.5 for the group treated with PEG-rVWF (MS EXCEL, trapezoidal model). Our study demonstrates that chemical modification by PEGylation of rVWF can be used to improve the survival of co-administered native rFVIII in the circulation of hemophilic mice.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V108.11.1002.1002
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 3
    Online Resource
    Online Resource
    Ethanol dependence of α 1 ‐antitrypsin C‐terminal Lys truncation mediated by basic carboxypeptidases
    Wiley ; 2008
    In:  Transfusion Vol. 48, No. 2 ( 2008-02), p. 314-320
    Details
    In: Transfusion, Wiley, Vol. 48, No. 2 ( 2008-02), p. 314-320
    Abstract: BACKGROUND: Patients with hereditary emphysema are treated with α 1 ‐antitrypsin (α 1 ‐proteinase inhibitor [A1PI]) concentrates. High‐resolution isoelectric focusing (IEF) analysis of A1PI shows that commercial A1PI products have different glycoisoform band patterns predominantly caused by varying degrees of C‐terminal Lys truncation at position 394 from the A1PI molecule. Basic carboxypeptidases (CPs) are a group of enzymes that specifically cleave C‐terminal basic amino acids (Arg or Lys) from peptides and proteins. STUDY DESIGN AND METHODS: In this study, whether A1PI is a substrate for basic CPs was investigated. CPN and CPU, two CPs present in plasma, and CPM, a GPI‐anchored membrane protein highly expressed in lung tissues, were included. RESULTS: Basic CPs are able to mediate the C‐terminal Lys truncation of A1PI although with a very low efficiency. However, presence of ethanol, for example, during Cohn fractionation, renders A1PI highly susceptible to cleavage by CP with the extent of Lys truncation depending on the ethanol concentration. This ethanol concentration dependence elegantly explains the varying amounts of des‐Lys A1PI present in commercial preparations purified from different Cohn fractions. CONCLUSIONS: The cause of C‐terminal truncation of A1PI present in products used for augmentation therapy has been identified, and it has been shown that A1PI becomes a substrate for CPs, specifically CPN, because of the presence of ethanol during Cohn fractionation.
    Type of Medium: Online Resource
    ISSN: 0041-1132 , 1537-2995
    URL: Issue
    URL: Article
    DOI: 10.1111/trf.2008.48.issue-2
    DOI: 10.1111/j.1537-2995.2007.01525.x
    Language: English
    Publisher: Wiley
    Publication Date: 2008
    detail.hit.zdb_id: 2018415-3
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  • 4
    Online Resource
    Online Resource
    Biochemical and Functional Characterization of PEGylated rVWF.
    American Society of Hematology ; 2006
    In:  Blood Vol. 108, No. 11 ( 2006-11-16), p. 1021-1021
    Details
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 1021-1021
    Abstract: Covalent modification of therapeutic proteins by polyethylene glycol derivatives is an established method for improving pharmacokinetic properties of therapeutic proteins. Highly purified rVWF expressed in CHO cells, was chemically modified via PEGylation of lysine residues at mild alkaline pH with PEG succinimidyl succinate (linear 5 kDa PEG). Increasing the amount of PEG used in the coupling procedure, the molecular size of VWF increased, as demonstrated in SDS-PAGE and by agarose gel electrophoreses indicating an increase in size of the bands resembling the VWF multimers. PEGylation of rVWF reduced platelet-aggregating and collagen-binding functions by 60% (VWF:RCo activity) and 40% (VWF:CB activity). While FVIII-binding capacity, measured by a FVIII binding ELISA, was reduced and reduction correlated with the amount of PEG bound to VWF, there was almost no effect on FVIII binding affinity which remained in the same order of magnitude as measured with non-PEGylated VWF. Similar results were obtained when rVWF was PEGylated via carbohydrate moieties after oxidation and subsequent derivatization with monomethoxy-PEG hydrazide. PEGylated rVWF was applied to VWF-deficient mice at a dose of 40 VWF:Ag U/kg and plasma levels were monitored for up to 24 hours. As a control, non-modified rVWF was applied to the animals. PEGylated VWF had substantially prolonged survival in the circulation compared with non-modified rVWF with an increase of the AUC by a factor of & gt;10. VWD mice substituted with human VWF show a secondary rise in FVIII bringing them into the FVIII levels measured in C57Bl/6 control mice. This secondary rise was sustained after treatment with PEGylated rVWF where FVIII levels above the starting level were measurable even 48 hours after injection while in the control group base line FVIII levels were reached already after 24 hours. rVWF is the largest protein ever PEGylated and PEGylation results in prolonged survival in the circulation while maintaining FVIII stabilizing functions of the VWF molecule in vivo.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V108.11.1021.1021
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 5
    Online Resource
    Online Resource
    Proteomic analysis of UVC irradiation-induced damage of plasma proteins: Serum amyloid P component as a major target of photolysis
    Wiley ; 2006
    In:  FEBS Letters Vol. 580, No. 13 ( 2006-05-29), p. 3229-3236
    Details
    In: FEBS Letters, Wiley, Vol. 580, No. 13 ( 2006-05-29), p. 3229-3236
    Type of Medium: Online Resource
    ISSN: 0014-5793
    URL: Article
    DOI: 10.1016/j.febslet.2006.05.002
    RVK:
    WA 15000
    Language: English
    Publisher: Wiley
    Publication Date: 2006
    detail.hit.zdb_id: 1460391-3
    SSG: 12
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  • 6
    Online Resource
    Online Resource
    Biochemical, molecular characterization, and glycoproteomic analyses of α 1 ‐proteinase inhibitor products used for replacement therapy*
    Wiley ; 2006
    In:  Transfusion Vol. 46, No. 11 ( 2006-11), p. 1959-1977
    Details
    In: Transfusion, Wiley, Vol. 46, No. 11 ( 2006-11), p. 1959-1977
    Abstract: BACKGROUND: Isoelectric focusing (IEF) of α 1 ‐proteinase inhibitor (A1PI) shows that commercial products and plasma have different glycoisoform band patterns. Those in Aralast (Grifols Biologicals) reflect an anodal shift of glycoisoforms, which has caused concern. The protein, including glycoproteomic analyses, and structural features of A1PI products were investigated by state‐of‐the‐art techniques. STUDY DESIGN AND METHODS: Batches from Aralast, Prolastin (Bayer), and Zemaira (Aventis Behring LLC) were analyzed by high‐resolution IEF and high‐performance size‐exclusion chromatography (HP‐SEC). Preparative separated isoforms from IEF were further purified by chromatography and subjected to mass spectrometry for sequence analyses, peptide mapping, and glycosylation analysis. Deamidation was quantified by enzymatic isoaspartate detection. Multiple sequence alignments and structural bioinformatics analyses were performed. RESULTS: In HP‐SEC, Prolastin had the highest aggregate content at approximately 30 percent. Isoforms from all products purified by high‐resolution IEF were sequenced with an amino acid coverage of more than 98 percent. Deamidation of Asn116 and Asn314 in A1PI was to found to some extent in all products and confirmed quantitatively by enzymatic analysis. There were no signs of methionine oxidation. Cys232 was found to be cysteinylated in A1PI in Prolastin and Aralast as in plasma, but not in Zemaira. All products showed truncation of the C‐terminal lysine. Intact A1PI concentrates contained mainly diantennary, disialylated and smaller amounts of triantennary, trisialylated N‐glycans. The percentage of fucosylation was similar in all products. Site‐specific glycan analysis revealed bands M6 contained only diantennary glycans, whereas the more acidic bands M4 and M2 also carried triantennary structures. The most acidic isoforms, M2 in Prolastin and Zemaira and M0 in Aralast, additionally exhibited tetraantennary N‐glycans. CONCLUSION: Protein chemical characterization of A1PI showed that all A1PI products to some extent differ from A1PI circulating in human plasma. Bioinformatic analysis indicated that removal of C‐terminal Lys394 and cysteinylation of Cys232 are unlikely to affect structure and/or function of A1PI but cysteinylation may influence interaction between A1PI and its physiologic ligands. Aralast, Prolastin, and Zemaira contain the same set of N‐glycans in the same ratios as those in normal human plasma A1PI. Tri‐ and tetraantennary structures are responsible for the partitioning into IEF isoforms, with the migration shift of Aralast not being due to any difference in the N‐glycosylation, but to the partial loss of the C‐terminal lysine.
    Type of Medium: Online Resource
    ISSN: 0041-1132 , 1537-2995
    URL: Issue
    URL: Article
    DOI: 10.1111/trf.2006.46.issue-11
    DOI: 10.1111/j.1537-2995.2006.01004.x
    Language: English
    Publisher: Wiley
    Publication Date: 2006
    detail.hit.zdb_id: 2018415-3
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  • 7
    Online Resource
    Online Resource
    Development of BAX 826, a Polysialylated Full-Length rFVIII with Significantly Improved PK Properties
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 3536-3536
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 3536-3536
    Abstract: Post-translational glycosylation determines the pharmacodynamic and pharmacokinetic properties of therapeutic proteins. While desialylation of FVIII/VWF (asialo-FVIII/VWF) has a significantly reduced half-life compared to normal FVIII/VWF complex (Sodetz et al 1977), we introduced additional sialic acids on recombinant (r) FVIII to obtain the opposite: prolonged survival of rFVIII in the circulation. Additional sialic acid residues were introduced to rFVIII by covalently binding polysialic acid (PSA) to full length (FL) rFVIII. The resulting drug candidate BAX 826, polysialylated human rFVIII, is manufactured from octocog alfa which is expressed in Chinese Hamster Ovary Cells by a plasma/albumin free cell culture method and is the active substance in Baxalta´s licensed product ADVATE. The manufacturing process for BAX 826 comprises several steps, starting with the bulk drug substance (BDS) of the ADVATE process which is subjected to polysialylation, i.e. covalent attachment of PSA of an average molecular weight of 20 kDa to rFVIII. Polysialylation is followed by a sequence of chromatographic purification steps and concentration of the conjugate by an ultra-/diafiltration step leading to the pre-formulated BDS. Final formulation of the BDS includes a filling and lyophilization step to obtain the final drug product. The process described is suited to manufacture BAX 826 in large scale and showed a good batch to batch consistency, ensuring an equivalent product quality for each batch. BAX 826 was extensively structurally and functionally characterized. The methods used included reducing and non-reducing peptide mapping to determine amino acid sequence and post translational modifications, qualitative analysis of modification sites, SDS-PAGE and Western blot analysis, assessment of three dimensional structure similarities of BAX 826 and rFVIII, ADVATE, by Fourier-transformed infrared spectroscopy (FTIR), dynamic light scattering (DLS) and circular dichroism (CD). Functional characterization was performed by assessment of the kinetics of the assembly and activity of FIXa-FVIII (tenase) complex, determination of the rate of activation and inactivation of BAX 826 by thrombin, determination of overall hemostatic potency by a thrombin generation assay, measurement of the rate of inactivation of untreated or thrombin-activated BAX 826 by activated protein C, measurement of kinetics of the binding of BAX 826 to von Willebrand factor (VWF), to phospholipids, and to low-density lipoprotein (LDL)-receptor-related protein 1 (LRP1) by surface plasmon resonance spectroscopy. While BAX 826 retained full hemostatic functionality of FVIII as a co-factor of the tenase complex, and can thus be considered a fully active FVIII molecule, it was found to have a reduced binding to VWF and to LRP1. Together with the conception that VWF as FVIII's chaperone dictates the maximally achievable terminal half-life extension for FVIII reduced binding to VWF and FVIII's major clearance receptor LRP1likely explains the prolonged pharmacokinetic (PK) properties when comparing BAX 826 with unmodified FL rFVIII in animal models. In summary, BAX 826, a polysialylated rFVIII derivative, can be manufactured reproducibly without relevant changes to the protein structure characteristic for a functional FVIII molecule with high specific activity. Disclosures Turecek: Baxalta Innovations GmbH: Employment. Siekmann:Baxalta Innovations GmbH: Employment. Mitterer:Baxalta Innovations GmH: Employment. Graninger:Baxalta Innovations GmbH: Employment. Schrenk:Baxalta Innovations GmbH: Employment. Matthiessen:Baxalta Innovations GmbH: Employment. Rottensteiner:Baxalta Innovations GmbH: Employment. Hoellriegl:Baxalta Innovations GmbH: Employment. Putz:Baxalta Innovations GmbH: Employment. Schwarz:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.3536.3536
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 8
    Online Resource
    Online Resource
    Modification of rVWF with Polysialic Acid: Biochemical and Functional Characterization in Mice with VWD.
    American Society of Hematology ; 2006
    In:  Blood Vol. 108, No. 11 ( 2006-11-16), p. 1001-1001
    Details
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 1001-1001
    Abstract: Post-translational glycosylation determines the pharmacodynamic and pharmacokinetic properties of therapeutic proteins. While desialylation of FVIII/VWF (asialo-FVIII/VWF) has a significantly reduced half-life compared to normal FVIII/VWF complex (Sodetz et al 1977), we tried to introduce additional sialic acids on rVWF to obtain the opposite: prolonged survival of rVWF in the circulation. Additional sialic acid residues were introduced to rVWF by chemically attaching polysialic acid (PSA, colominic acid) to lysines on recombinant VWF. rVWF was purified from the cell culture supernatant of CHO cells co-expressing rFVIII and rVWF. Coupling of PSA was performed by terminal oxidation of the vicinal diols in PSA to aldehydes and binding to the primary amino groups in the VWF molecule by Schiff-base formation. Schiff-bases were stabilized by reduction with NaCNBH3 under formation of a secondary amine. PSA-rVWF conjugates retained their multimeric structure but had slightly increased molecular sizes of the VWF sub-units due to bound PSA. The attachment of PSA to rVWF resulted in a decrease of RCo activity and a decrease in FVIII binding capacity to approximately 60%. Administration of PSA-rVWF at a dose of 100 units rVWF antigen per kg to VWF-deficient mice resulted in a 2-fold increase in the survival of rVWF as compared to non-modified rVWF. A secondary rise in endogenous murine FVIII after substitution of VWD mice with human VWF occurred elevating FVIII levels from 0.1 (baseline) to 0.4 IU FVIII/mL at 3 hours after application of both non-modified and polysialylated rVWF. Maximum levels of ~ 0.4 IU FVIII/mL were maintained for more than 20 hours in the group treated with PSA-rVWF despite the reduced FVIII binding capacity observed in vitro, while FVIII levels in mice treated with non-modified rVWF steadily declined and returned to their baseline level after 21 hours. The FVIII AUC was 5.3 and 3.3 IU/mL/hr for mice treated with PSA-rVWF and non-modified rVWF, respectively. We conclude that polysialylation of rVWF represents a method to prolong the survival of rVWF in the circulation.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V108.11.1001.1001
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 9
    Online Resource
    Online Resource
    Neuroscience data and tool sharing : A legal and policy framework for neuroinformatics
    Springer Science and Business Media LLC ; 2003
    In:  Neuroinformatics Vol. 1, No. 2 ( 2003-6), p. 149-165
    Details
    In: Neuroinformatics, Springer Science and Business Media LLC, Vol. 1, No. 2 ( 2003-6), p. 149-165
    Type of Medium: Online Resource
    ISSN: 1539-2791 , 1559-0089
    URL: Article
    DOI: 10.1007/s12021-003-0002-1
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2003
    detail.hit.zdb_id: 2099780-2
    SSG: 12
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  • 10
    Online Resource
    Online Resource
    Verwendung komplementärmedizinischer Verfahren im Alter
    Georg Thieme Verlag KG ; 2010
    In:  Deutsche Zeitschrift für Onkologie Vol. 42, No. 01 ( 2010-3), p. 6-11
    Details
    In: Deutsche Zeitschrift für Onkologie, Georg Thieme Verlag KG, Vol. 42, No. 01 ( 2010-3), p. 6-11
    Type of Medium: Online Resource
    ISSN: 1617-5891 , 1439-0930
    URL: Article
    DOI: 10.1055/s-0029-1242582
    Language: German
    Publisher: Georg Thieme Verlag KG
    Publication Date: 2010
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