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Identification of Subvisible Particles in Biopharmaceutical Formulations Using Raman Spectroscopy Provides Insight into Polysorbate 20 Degradation Pathway.

Saggu M, Liu J, Patel A - Pharm. Res. (2015)

Bottom Line: To study composition and heterogeneity of insoluble subvisible particles in Mab formulations resulting from degradation of polysorbate 20 and to develop a better understanding of the mechanisms of polysorbate degradation leading to particle formation.Most of the subvisible particles identified were comprised of mixtures of fatty acids with no observable signal from fatty acid esters consistent with hydrolysis being the predominant degradation mechanism leading to particulate formation under these storage conditions.Our methodology is generally applicable for identification of particles in antibody formulations and, in particular, has the potential to give detailed information about particle heterogeneity and insight into mechanistic aspects of particle formation.

View Article: PubMed Central - PubMed

Affiliation: Late Stage Pharmaceutical Development, Genentech Inc., South San Francisco, California, 94080, USA, saggu.miguel@gene.com.

ABSTRACT

Purpose: To study composition and heterogeneity of insoluble subvisible particles in Mab formulations resulting from degradation of polysorbate 20 and to develop a better understanding of the mechanisms of polysorbate degradation leading to particle formation.

Methods: In this study, we exploit the potential of Raman microscopy for chemical identification of particles in monoclonal antibody formulations. Through a combination of experiments and density functional theory (DFT) calculations, we identified unique spectral marker bands for insoluble degradation products of polysorbate 20. We first applied our methodology to identify particles in model systems containing complex mixtures of fatty acids and then to subvisible particles in antibody formulations stored at 5°C for several years.

Results: Most of the subvisible particles identified were comprised of mixtures of fatty acids with no observable signal from fatty acid esters consistent with hydrolysis being the predominant degradation mechanism leading to particulate formation under these storage conditions.

Conclusions: Our methodology is generally applicable for identification of particles in antibody formulations and, in particular, has the potential to give detailed information about particle heterogeneity and insight into mechanistic aspects of particle formation.

No MeSH data available.


Experimental Raman spectra of fatty acid particles in model systems of polysorbate 20 digested with (a) rabbit liver esterase and (b) pancreatic lipase. Experimental conditions: T = 233 K, 16 mW laser power, 300 s accumulation time. For comparison the reference spectra of capric, lauric, myristic and palmitic acid are shown.
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Fig4: Experimental Raman spectra of fatty acid particles in model systems of polysorbate 20 digested with (a) rabbit liver esterase and (b) pancreatic lipase. Experimental conditions: T = 233 K, 16 mW laser power, 300 s accumulation time. For comparison the reference spectra of capric, lauric, myristic and palmitic acid are shown.

Mentions: The spectra for several particles highlighting the range between 1000 and 1200 cm−1 are shown in Fig. 4A. The spectra showing the entire spectral range can be found in Fig. S6. It can be seen that the two strong bands arising from ν(C-C) stretches at 1065 and 1126 cm−1 are the same in all particles and do not allow for discrimination of different fatty acids. However, the marker band of the unique ν(C-C) stretch located between those two bands shows heterogeneity with main peaks at both 1085 and 1093 cm−1 in agreement with the unique ν(C-C) stretch vibrations found in lauric and myristic acid. No band at 1175 cm−1 is present indicating either the absence of capric acid or the possibility that most of the capric acid is still solubilized and not a constituent of the particles. Some particles show a small shoulder around 1100 cm−1 suggesting the presence of small amounts of palmitic acid. The ratio between lauric and myristic acid is ~1:1. Based on the relative abundance of lauric and myristic acid in polysorbate 20 as describend above (3.5:1 ratio between lauric and myristic acid) this can be interpreted in terms of their different solubility (short chain fatty acids have a higher solubility). So even though there will be more lauric acid side chains in the polysorbate 20 raw material it is likely that a larger fraction of it is solubilized and that the actual insoluble particles have larger contributions of the longer chain fatty acids. In conclusion, most insoluble particles on the filter are mixtures of lauric acid, myristic acid, and possibly small amounts of palmitic acid.Fig. 4


Identification of Subvisible Particles in Biopharmaceutical Formulations Using Raman Spectroscopy Provides Insight into Polysorbate 20 Degradation Pathway.

Saggu M, Liu J, Patel A - Pharm. Res. (2015)

Experimental Raman spectra of fatty acid particles in model systems of polysorbate 20 digested with (a) rabbit liver esterase and (b) pancreatic lipase. Experimental conditions: T = 233 K, 16 mW laser power, 300 s accumulation time. For comparison the reference spectra of capric, lauric, myristic and palmitic acid are shown.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4526581&req=5

Fig4: Experimental Raman spectra of fatty acid particles in model systems of polysorbate 20 digested with (a) rabbit liver esterase and (b) pancreatic lipase. Experimental conditions: T = 233 K, 16 mW laser power, 300 s accumulation time. For comparison the reference spectra of capric, lauric, myristic and palmitic acid are shown.
Mentions: The spectra for several particles highlighting the range between 1000 and 1200 cm−1 are shown in Fig. 4A. The spectra showing the entire spectral range can be found in Fig. S6. It can be seen that the two strong bands arising from ν(C-C) stretches at 1065 and 1126 cm−1 are the same in all particles and do not allow for discrimination of different fatty acids. However, the marker band of the unique ν(C-C) stretch located between those two bands shows heterogeneity with main peaks at both 1085 and 1093 cm−1 in agreement with the unique ν(C-C) stretch vibrations found in lauric and myristic acid. No band at 1175 cm−1 is present indicating either the absence of capric acid or the possibility that most of the capric acid is still solubilized and not a constituent of the particles. Some particles show a small shoulder around 1100 cm−1 suggesting the presence of small amounts of palmitic acid. The ratio between lauric and myristic acid is ~1:1. Based on the relative abundance of lauric and myristic acid in polysorbate 20 as describend above (3.5:1 ratio between lauric and myristic acid) this can be interpreted in terms of their different solubility (short chain fatty acids have a higher solubility). So even though there will be more lauric acid side chains in the polysorbate 20 raw material it is likely that a larger fraction of it is solubilized and that the actual insoluble particles have larger contributions of the longer chain fatty acids. In conclusion, most insoluble particles on the filter are mixtures of lauric acid, myristic acid, and possibly small amounts of palmitic acid.Fig. 4

Bottom Line: To study composition and heterogeneity of insoluble subvisible particles in Mab formulations resulting from degradation of polysorbate 20 and to develop a better understanding of the mechanisms of polysorbate degradation leading to particle formation.Most of the subvisible particles identified were comprised of mixtures of fatty acids with no observable signal from fatty acid esters consistent with hydrolysis being the predominant degradation mechanism leading to particulate formation under these storage conditions.Our methodology is generally applicable for identification of particles in antibody formulations and, in particular, has the potential to give detailed information about particle heterogeneity and insight into mechanistic aspects of particle formation.

View Article: PubMed Central - PubMed

Affiliation: Late Stage Pharmaceutical Development, Genentech Inc., South San Francisco, California, 94080, USA, saggu.miguel@gene.com.

ABSTRACT

Purpose: To study composition and heterogeneity of insoluble subvisible particles in Mab formulations resulting from degradation of polysorbate 20 and to develop a better understanding of the mechanisms of polysorbate degradation leading to particle formation.

Methods: In this study, we exploit the potential of Raman microscopy for chemical identification of particles in monoclonal antibody formulations. Through a combination of experiments and density functional theory (DFT) calculations, we identified unique spectral marker bands for insoluble degradation products of polysorbate 20. We first applied our methodology to identify particles in model systems containing complex mixtures of fatty acids and then to subvisible particles in antibody formulations stored at 5°C for several years.

Results: Most of the subvisible particles identified were comprised of mixtures of fatty acids with no observable signal from fatty acid esters consistent with hydrolysis being the predominant degradation mechanism leading to particulate formation under these storage conditions.

Conclusions: Our methodology is generally applicable for identification of particles in antibody formulations and, in particular, has the potential to give detailed information about particle heterogeneity and insight into mechanistic aspects of particle formation.

No MeSH data available.