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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.


Raman spectra of common particulates and compounds found in biopharmaceutical formulations (a) lauric acid (b) ethylene glycol monolaurate (c) mechanically stressed antibody (d) silicone oil (e) cellulose and (f) polypropylene. Experimental conditions: 4 mW laser power, T = 298 K, 60 s accumulation time.
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Fig3: Raman spectra of common particulates and compounds found in biopharmaceutical formulations (a) lauric acid (b) ethylene glycol monolaurate (c) mechanically stressed antibody (d) silicone oil (e) cellulose and (f) polypropylene. Experimental conditions: 4 mW laser power, T = 298 K, 60 s accumulation time.

Mentions: This section describes the general advantage of Raman spectroscopy to distinguish between intrinsic, extrinsic as well as inherent particles. Most particles have distinct spectral features allowing for unambiguous identification (see below). Figure 3 shows Raman spectra of particles, which are likely to be found in antibody formulations. For comparison, trace (a) contains the spectrum of dry lauric acid, which is a degradation product of polysorbate 20 resulting from hydrolysis of the side chain. The spectral features have already been discussed in the previous section. Trace (b) shows the Raman spectrum of ethylene glycol monolaurate, which is one of the expected degradation products of polysorbate 20 caused by oxidation. Qualitatively, this spectrum is very similar to lauric acid. However, subtle differences make it possible to differentiate between both of them. The ν(C = O) stretch vibration is blue-shifted to 1740 cm−1 and has a small linewidth of 10 cm−1 indicating that the carbonyl group is not hydrogen bonding in the crystal structure in ethylene glycol monolaurate. The δ(CH2) rocking vibrations at 893 and 908 cm−1 are merged together into one broadened band at 890 cm−1. The most prominent differences are changes in these δ(CH2) rocking modes, which are red-shifted to 890 and 885 cm−1, and the absence of the characteristic low-frequency vibration in ethylene glycol monolaurate (466 cm−1 in lauric acid). Another unique feature of ethylene glycol monolaurate is the presence of a medium intensity band at 1158 cm−1, which can be attributed to a ν(C-C) stretch vibration. For comparison, the Raman spectrum of ethylene glycol monolaurate was calculated using DFT as well (Fig. S2). The most prominent differences to lauric acid are captured by the calculations as well, i.e., changes in the rocking modes as well as the disappearance of the low-frequency vibration. There is very good agreement between the experimental and calculated carbonyl stretch vibration ν(C = O) further corroborating the experimental finding that the carbonyl group of ethylene glycol monolaurate is not hydrogen bonded in the crystal structure.Fig. 3


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)

Raman spectra of common particulates and compounds found in biopharmaceutical formulations (a) lauric acid (b) ethylene glycol monolaurate (c) mechanically stressed antibody (d) silicone oil (e) cellulose and (f) polypropylene. Experimental conditions: 4 mW laser power, T = 298 K, 60 s accumulation time.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: Raman spectra of common particulates and compounds found in biopharmaceutical formulations (a) lauric acid (b) ethylene glycol monolaurate (c) mechanically stressed antibody (d) silicone oil (e) cellulose and (f) polypropylene. Experimental conditions: 4 mW laser power, T = 298 K, 60 s accumulation time.
Mentions: This section describes the general advantage of Raman spectroscopy to distinguish between intrinsic, extrinsic as well as inherent particles. Most particles have distinct spectral features allowing for unambiguous identification (see below). Figure 3 shows Raman spectra of particles, which are likely to be found in antibody formulations. For comparison, trace (a) contains the spectrum of dry lauric acid, which is a degradation product of polysorbate 20 resulting from hydrolysis of the side chain. The spectral features have already been discussed in the previous section. Trace (b) shows the Raman spectrum of ethylene glycol monolaurate, which is one of the expected degradation products of polysorbate 20 caused by oxidation. Qualitatively, this spectrum is very similar to lauric acid. However, subtle differences make it possible to differentiate between both of them. The ν(C = O) stretch vibration is blue-shifted to 1740 cm−1 and has a small linewidth of 10 cm−1 indicating that the carbonyl group is not hydrogen bonding in the crystal structure in ethylene glycol monolaurate. The δ(CH2) rocking vibrations at 893 and 908 cm−1 are merged together into one broadened band at 890 cm−1. The most prominent differences are changes in these δ(CH2) rocking modes, which are red-shifted to 890 and 885 cm−1, and the absence of the characteristic low-frequency vibration in ethylene glycol monolaurate (466 cm−1 in lauric acid). Another unique feature of ethylene glycol monolaurate is the presence of a medium intensity band at 1158 cm−1, which can be attributed to a ν(C-C) stretch vibration. For comparison, the Raman spectrum of ethylene glycol monolaurate was calculated using DFT as well (Fig. S2). The most prominent differences to lauric acid are captured by the calculations as well, i.e., changes in the rocking modes as well as the disappearance of the low-frequency vibration. There is very good agreement between the experimental and calculated carbonyl stretch vibration ν(C = O) further corroborating the experimental finding that the carbonyl group of ethylene glycol monolaurate is not hydrogen bonded in the crystal structure.Fig. 3

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.