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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 isolated particles from Mab1 and Mab2 formulations. For comparison the reference spectra of capric, lauric, myristic, palmitic acid and ethylene glycol monolaurate (EGM) are shown. Experimental conditions: (a) T = 233 K, 16 mW laser power, 300 s accumulation time. Particle 1 and 2 were transferred directly to the cold Raman cell. Particles 3–5 were dried over phosphorous pentoxide for 2 h before measurement (b) T = −40°C, 4 mW laser power, 90 s accumulation time. Particles were dried over phosphorous pentoxide for 2 h before measurement.
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Fig5: Experimental Raman spectra of isolated particles from Mab1 and Mab2 formulations. For comparison the reference spectra of capric, lauric, myristic, palmitic acid and ethylene glycol monolaurate (EGM) are shown. Experimental conditions: (a) T = 233 K, 16 mW laser power, 300 s accumulation time. Particle 1 and 2 were transferred directly to the cold Raman cell. Particles 3–5 were dried over phosphorous pentoxide for 2 h before measurement (b) T = −40°C, 4 mW laser power, 90 s accumulation time. Particles were dried over phosphorous pentoxide for 2 h before measurement.

Mentions: Particles from Mab2 formulation were dried over phosphorous pentoxide for 2 h before measurement. The spectra are similar to the ones obtained from particles in Mab1 formulation (Fig. 5b). The chemical identity of the particles is comprised mainly of palmitic and myristic acid. In a control experiment, particles from Mab2 formulation were dried overnight over phosphorus pentoxide. After drying, partial recovery of the low-frequency vibration at 376 cm−1 was observed consistent with the presence of palmitic acid, which showed similar behavior (Fig. S8). No further recovery was achieved by longer drying times. Unfortunately, drying under these conditions leads to loss of short chain fatty acids due to their higher volatility. Therefore, we do not recommend prolonged drying of particles. To recover spectra identical to the library spectra shown in Fig. 1, it may be necessary to perform a temperature jump and recrystallize the fatty acids back to their original crystal structure.Fig. 5


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 isolated particles from Mab1 and Mab2 formulations. For comparison the reference spectra of capric, lauric, myristic, palmitic acid and ethylene glycol monolaurate (EGM) are shown. Experimental conditions: (a) T = 233 K, 16 mW laser power, 300 s accumulation time. Particle 1 and 2 were transferred directly to the cold Raman cell. Particles 3–5 were dried over phosphorous pentoxide for 2 h before measurement (b) T = −40°C, 4 mW laser power, 90 s accumulation time. Particles were dried over phosphorous pentoxide for 2 h before measurement.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526581&req=5

Fig5: Experimental Raman spectra of isolated particles from Mab1 and Mab2 formulations. For comparison the reference spectra of capric, lauric, myristic, palmitic acid and ethylene glycol monolaurate (EGM) are shown. Experimental conditions: (a) T = 233 K, 16 mW laser power, 300 s accumulation time. Particle 1 and 2 were transferred directly to the cold Raman cell. Particles 3–5 were dried over phosphorous pentoxide for 2 h before measurement (b) T = −40°C, 4 mW laser power, 90 s accumulation time. Particles were dried over phosphorous pentoxide for 2 h before measurement.
Mentions: Particles from Mab2 formulation were dried over phosphorous pentoxide for 2 h before measurement. The spectra are similar to the ones obtained from particles in Mab1 formulation (Fig. 5b). The chemical identity of the particles is comprised mainly of palmitic and myristic acid. In a control experiment, particles from Mab2 formulation were dried overnight over phosphorus pentoxide. After drying, partial recovery of the low-frequency vibration at 376 cm−1 was observed consistent with the presence of palmitic acid, which showed similar behavior (Fig. S8). No further recovery was achieved by longer drying times. Unfortunately, drying under these conditions leads to loss of short chain fatty acids due to their higher volatility. Therefore, we do not recommend prolonged drying of particles. To recover spectra identical to the library spectra shown in Fig. 1, it may be necessary to perform a temperature jump and recrystallize the fatty acids back to their original crystal structure.Fig. 5

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.