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Understanding the relationship between biotherapeutic protein stability and solid-liquid interfacial shear in constant region mutants of IgG1 and IgG4.

Tavakoli-Keshe R, Phillips JJ, Turner R, Bracewell DG - J Pharm Sci (2013)

Bottom Line: Results suggest that the techniques are orthogonal, with thermal methods based on intramolecular interaction and shear device stability based on localized unfolding revealing less stable regions that drive aggregation.Molecular modeling shows the modifications' effects on the antibody structures and indicates a possible role for Fc conformation and Fab-Fc docking in determining suspended protein stability.The data introduce the PDC value as an orthogonal stability indicator, complementary to traditional thermal methods, allowing lead antibody selection based on a more full understanding of process stability.

View Article: PubMed Central - PubMed

Affiliation: The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK; MedImmune, Granta Park, Cambridge, CB21 6GH, UK.

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Comparison of HPLC traces for 5 mg/mL IgG1 WT samples processed at different conditions. (a) ____ Before processing, (b) —– after 24 h hold at pH 4 and 40°C, (c) after 60 min shear at 9000 rpm.
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fig06: Comparison of HPLC traces for 5 mg/mL IgG1 WT samples processed at different conditions. (a) ____ Before processing, (b) —– after 24 h hold at pH 4 and 40°C, (c) after 60 min shear at 9000 rpm.

Mentions: HPLC traces from samples aggregated through the interfacial shear device were compared with those that had been degraded through thermal routes (Fig. 6). Interfacial shear-device-degraded samples showed a reduction of monomer and no measurable increase in fragments or multimers. This indicates that the antibody had aggregated and formed much larger particles that had begun to fall out of solution, as shown by the increased opaqueness of samples taken from the device. This implies that the multimers are either very short lived, making them difficult to detect, or they are formed at the solid–liquid interface prior to release into the bulk solution. Thermally denatured samples showed a reduction of monomer, with the majority of the antibody found as low-molecular-weight multimer, suggesting a more classically observed nucleation mechanism for the aggregation.15


Understanding the relationship between biotherapeutic protein stability and solid-liquid interfacial shear in constant region mutants of IgG1 and IgG4.

Tavakoli-Keshe R, Phillips JJ, Turner R, Bracewell DG - J Pharm Sci (2013)

Comparison of HPLC traces for 5 mg/mL IgG1 WT samples processed at different conditions. (a) ____ Before processing, (b) —– after 24 h hold at pH 4 and 40°C, (c) after 60 min shear at 9000 rpm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263191&req=5

fig06: Comparison of HPLC traces for 5 mg/mL IgG1 WT samples processed at different conditions. (a) ____ Before processing, (b) —– after 24 h hold at pH 4 and 40°C, (c) after 60 min shear at 9000 rpm.
Mentions: HPLC traces from samples aggregated through the interfacial shear device were compared with those that had been degraded through thermal routes (Fig. 6). Interfacial shear-device-degraded samples showed a reduction of monomer and no measurable increase in fragments or multimers. This indicates that the antibody had aggregated and formed much larger particles that had begun to fall out of solution, as shown by the increased opaqueness of samples taken from the device. This implies that the multimers are either very short lived, making them difficult to detect, or they are formed at the solid–liquid interface prior to release into the bulk solution. Thermally denatured samples showed a reduction of monomer, with the majority of the antibody found as low-molecular-weight multimer, suggesting a more classically observed nucleation mechanism for the aggregation.15

Bottom Line: Results suggest that the techniques are orthogonal, with thermal methods based on intramolecular interaction and shear device stability based on localized unfolding revealing less stable regions that drive aggregation.Molecular modeling shows the modifications' effects on the antibody structures and indicates a possible role for Fc conformation and Fab-Fc docking in determining suspended protein stability.The data introduce the PDC value as an orthogonal stability indicator, complementary to traditional thermal methods, allowing lead antibody selection based on a more full understanding of process stability.

View Article: PubMed Central - PubMed

Affiliation: The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK; MedImmune, Granta Park, Cambridge, CB21 6GH, UK.

Show MeSH
Related in: MedlinePlus