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Evaluation of Immunostimulatory Potential of Branded and US-Generic Enoxaparins in an In Vitro Human Immune System Model.

Luna E, Agrawal P, Mehta R, Vernhes C, Viskov C, Amiral J, Warren WL, Drake DR - Clin. Appl. Thromb. Hemost. (2014)

Bottom Line: Low-molecular-weight heparins (LMWHs) have several positive therapeutic effects and can also form immunostimulatory complexes with plasma proteins, such as platelet factor 4 (PF4).Production of tissue factor pathway inhibitor (TFPI), a physiologic heparin-induced inhibitor of tissue factor-induced coagulation that was used as a functional readout of biological activity of enoxaparins in these assays, was heightened in the presence of branded enoxaparin complexes, but its levels were variable in cultures treated with complexes containing US-generic enoxaparins.Analytical analyses suggest that the heightened immunostimulatory potential of some of the US-generic enoxaparin product lots could be tied to their capacity to form ultra-large and/or more stable complexes with PF4 than the other LMWHs included in this study.

View Article: PubMed Central - PubMed

Affiliation: Sanofi Pasteur, VaxDesign Campus, Orlando, FL, USA.

No MeSH data available.


Related in: MedlinePlus

Demonstration of differential release of PF4 from complexes formed with branded and US-generic enoxaparins. A, Chromatogram of SE-FPLC analysis of PF4 alone and all Lot 1, Batch 1 PF4–enoxaparin complexes performed at 0.15 mol/L NaCl (upper panel) and 0.75 mol/L NaCl (lower panel). B, 4% to 20% gradient native PAGE gel analysis of PF4–enoxaparin complexes under nonreducing conditions followed by silver staining to resolve the protein bands. These data are representative of 3 independent analyses. ELISA indicates enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; PF4, platelet factor 4; PF4–H, PF4–heparin complex; SD, standard deviation; SE-FPLC, size exclusion-fast performance liquid chromatography.
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fig6-1076029614562037: Demonstration of differential release of PF4 from complexes formed with branded and US-generic enoxaparins. A, Chromatogram of SE-FPLC analysis of PF4 alone and all Lot 1, Batch 1 PF4–enoxaparin complexes performed at 0.15 mol/L NaCl (upper panel) and 0.75 mol/L NaCl (lower panel). B, 4% to 20% gradient native PAGE gel analysis of PF4–enoxaparin complexes under nonreducing conditions followed by silver staining to resolve the protein bands. These data are representative of 3 independent analyses. ELISA indicates enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; PF4, platelet factor 4; PF4–H, PF4–heparin complex; SD, standard deviation; SE-FPLC, size exclusion-fast performance liquid chromatography.

Mentions: We considered the possibility that the differential immunostimulatory potential of PF4 complexes prepared with branded and US-generic enoxaparins may have resulted from possible differences in the physicochemical properties of these macromolecules. To test this theory, the size and strength of intramolecular interactions of blinded study (Lot 1, Batch 1) complexes were tested via SE-FPLC analysis under physiologic (0.15 mol/L) and high (0.75 mol/L) NaCl concentrations. Human PF4 without heparin, which was used to establish a baseline reading in the chromatography assay, generated 2 peaks (principally monomers and dimers) that eluted at graph positions 11 to 13 under physiologic (0.15 mol/L) NaCl conditions (Figure 6A, upper row). At a high (0.75 mol/L) salt concentration, PF4 was shown to be homogeneous and in the tetrameric form, that is, an elution peak at position ∼10 on the graph (Figure 6A, bottom row). This result is consistent with prior studies showing that a high salt concentration favors the formation of tetrameric PF4 molecules.13 When PF4 was complexed with branded enoxaparin, it generated a single peak that eluted at graph position ∼6 under physiologic salt concentrations (Figure 6A, upper row), which suggests the branded product forms homogenous complexes smaller than the 500-kDa molecular weight cutoff of the column. These complexes also appeared to readily dissociate from the polysaccharides in the high NaCl concentration buffer, yielding a major peak of tetrameric PF4 that eluted at position ∼10 on the graph (Figure 6A, bottom row). The US-generic enoxaparins yielded quite a distinct SE-FPLC profile. First, the complexes failed to generate peaks under physiologic salt conditions, which suggest the complexes were larger than the 500-kDa molecular weight cutoff of the column (Figure 6A, upper row). Second, these complexes appeared to poorly dissociate under high salt concentrations, since there were only minor PF4 peaks at the graph elution position of ∼10 in the Amphastar and Sandoz conditions (Figure 6A, bottom row).


Evaluation of Immunostimulatory Potential of Branded and US-Generic Enoxaparins in an In Vitro Human Immune System Model.

Luna E, Agrawal P, Mehta R, Vernhes C, Viskov C, Amiral J, Warren WL, Drake DR - Clin. Appl. Thromb. Hemost. (2014)

Demonstration of differential release of PF4 from complexes formed with branded and US-generic enoxaparins. A, Chromatogram of SE-FPLC analysis of PF4 alone and all Lot 1, Batch 1 PF4–enoxaparin complexes performed at 0.15 mol/L NaCl (upper panel) and 0.75 mol/L NaCl (lower panel). B, 4% to 20% gradient native PAGE gel analysis of PF4–enoxaparin complexes under nonreducing conditions followed by silver staining to resolve the protein bands. These data are representative of 3 independent analyses. ELISA indicates enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; PF4, platelet factor 4; PF4–H, PF4–heparin complex; SD, standard deviation; SE-FPLC, size exclusion-fast performance liquid chromatography.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC4401814&req=5

fig6-1076029614562037: Demonstration of differential release of PF4 from complexes formed with branded and US-generic enoxaparins. A, Chromatogram of SE-FPLC analysis of PF4 alone and all Lot 1, Batch 1 PF4–enoxaparin complexes performed at 0.15 mol/L NaCl (upper panel) and 0.75 mol/L NaCl (lower panel). B, 4% to 20% gradient native PAGE gel analysis of PF4–enoxaparin complexes under nonreducing conditions followed by silver staining to resolve the protein bands. These data are representative of 3 independent analyses. ELISA indicates enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; PF4, platelet factor 4; PF4–H, PF4–heparin complex; SD, standard deviation; SE-FPLC, size exclusion-fast performance liquid chromatography.
Mentions: We considered the possibility that the differential immunostimulatory potential of PF4 complexes prepared with branded and US-generic enoxaparins may have resulted from possible differences in the physicochemical properties of these macromolecules. To test this theory, the size and strength of intramolecular interactions of blinded study (Lot 1, Batch 1) complexes were tested via SE-FPLC analysis under physiologic (0.15 mol/L) and high (0.75 mol/L) NaCl concentrations. Human PF4 without heparin, which was used to establish a baseline reading in the chromatography assay, generated 2 peaks (principally monomers and dimers) that eluted at graph positions 11 to 13 under physiologic (0.15 mol/L) NaCl conditions (Figure 6A, upper row). At a high (0.75 mol/L) salt concentration, PF4 was shown to be homogeneous and in the tetrameric form, that is, an elution peak at position ∼10 on the graph (Figure 6A, bottom row). This result is consistent with prior studies showing that a high salt concentration favors the formation of tetrameric PF4 molecules.13 When PF4 was complexed with branded enoxaparin, it generated a single peak that eluted at graph position ∼6 under physiologic salt concentrations (Figure 6A, upper row), which suggests the branded product forms homogenous complexes smaller than the 500-kDa molecular weight cutoff of the column. These complexes also appeared to readily dissociate from the polysaccharides in the high NaCl concentration buffer, yielding a major peak of tetrameric PF4 that eluted at position ∼10 on the graph (Figure 6A, bottom row). The US-generic enoxaparins yielded quite a distinct SE-FPLC profile. First, the complexes failed to generate peaks under physiologic salt conditions, which suggest the complexes were larger than the 500-kDa molecular weight cutoff of the column (Figure 6A, upper row). Second, these complexes appeared to poorly dissociate under high salt concentrations, since there were only minor PF4 peaks at the graph elution position of ∼10 in the Amphastar and Sandoz conditions (Figure 6A, bottom row).

Bottom Line: Low-molecular-weight heparins (LMWHs) have several positive therapeutic effects and can also form immunostimulatory complexes with plasma proteins, such as platelet factor 4 (PF4).Production of tissue factor pathway inhibitor (TFPI), a physiologic heparin-induced inhibitor of tissue factor-induced coagulation that was used as a functional readout of biological activity of enoxaparins in these assays, was heightened in the presence of branded enoxaparin complexes, but its levels were variable in cultures treated with complexes containing US-generic enoxaparins.Analytical analyses suggest that the heightened immunostimulatory potential of some of the US-generic enoxaparin product lots could be tied to their capacity to form ultra-large and/or more stable complexes with PF4 than the other LMWHs included in this study.

View Article: PubMed Central - PubMed

Affiliation: Sanofi Pasteur, VaxDesign Campus, Orlando, FL, USA.

No MeSH data available.


Related in: MedlinePlus