Comparison of disaccharides and polyalcohols as stabilizers in freeze-dried protein formulations

Petteri Heljo

Research output: ThesisDoctoral ThesisCollection of Articles

Abstract

Protein-structured pharmaceuticals have become very important for treating several chronic and acute illnesses, but their commercialization is sometimes impeded by their poor stability in aqueous solutions. In some cases, protein stability may be better in dry than in aqueous formulations, and freeze drying (lyophilization) is the most commonly used method for producing such products. However, excipients are needed to keep the proteins from degrading during processing and storage, and the current selection of stabilizing compounds accepted by the regulatory authorities is limited. The aim of this thesis was to compare the stabilizing efficacies of different disaccharides and polyalcohols in protein formulations.

Firstly, the efficacies of 11 disaccharides and polyalcohols as β-galactosidase-protecting compounds were characterized at three different concentrations. Disaccharides were generally more effective than polyalcohols, which appeared to be caused in some cases by their lower crystallization tendency. Four disaccharides (cellobiose, melibiose, sucrose and trehalose) were selected for further secondary structure stabilization studies, which corroborated the results of the activity analysis studies that the protein-protecting efficacies of trehalose and melibiose differed from those of sucrose and cellobiose.

Secondly, the water plasticization and crystallization properties of the four disaccharides were studied after freeze drying by storing them at different relative humidity atmospheres. Cellobiose and sucrose were shown to crystallize faster and at lower relative humidity than trehalose and melibiose. The water plasticization properties of sucrose were shown to differ from the other disaccharides. Melibiose was most stable of the four compounds in amorphous state, which may have partly been caused by its slower molecular relaxation rate compared to trehalose.

Thirdly, the effect of freeze drying parameters on polyclonal IgG stability was studied in order to find out how much the stability of such antibodies could be affected by processing conditions in formulations containing trehalose. While varying the primary drying pressure and secondary drying heating rate did not directly affect protein stability, increased aggregation did occur at lower primary drying pressures when sodium phosphate buffer was used in the formulation. This may have been caused by buffer freeze-concentration, which may have been more significant when the sample temperature was lowered below the glass transition temperature of the freeze-concentrated phase. Sodium chloride was also shown to reduce the Z-average diameter of the rehydrated polyclonal IgG lyophilizates.

Finally, the efficacies of trehalose and melibiose in protecting a monoclonal antibody (rituximab) from degradation during freeze drying and storage at different relative humidity atmospheres were compared by storing the lyophilizates up to 3 months. Melibiose was generally more effective than trehalose in inhibiting secondary structure alterations, fragmentation and aggregation during drying and storage. Storage at dry conditions (RH 5%) was shown to be more harmful for rituximab in the lyophilized formulations than storage at more humid conditions (RH 11-23%), which may have been caused by the susceptibility of the protein to drying-induced degradation.
Original languageEnglish
Place of PublicationHelsinki
Publisher
Print ISBNs978-952-10-8616-8
Electronic ISBNs978-952-10-8617-5
Publication statusPublished - 2013
MoE publication typeG5 Doctoral dissertation (article)

Fields of Science

  • 317 Pharmacy

Cite this

Heljo, Petteri. / Comparison of disaccharides and polyalcohols as stabilizers in freeze-dried protein formulations. Helsinki : University of Helsinki, 2013. 64 p.
@phdthesis{5f8bc650b86a4f6bbe09ad57bc074b93,
title = "Comparison of disaccharides and polyalcohols as stabilizers in freeze-dried protein formulations",
abstract = "Protein-structured pharmaceuticals have become very important for treating several chronic and acute illnesses, but their commercialization is sometimes impeded by their poor stability in aqueous solutions. In some cases, protein stability may be better in dry than in aqueous formulations, and freeze drying (lyophilization) is the most commonly used method for producing such products. However, excipients are needed to keep the proteins from degrading during processing and storage, and the current selection of stabilizing compounds accepted by the regulatory authorities is limited. The aim of this thesis was to compare the stabilizing efficacies of different disaccharides and polyalcohols in protein formulations.Firstly, the efficacies of 11 disaccharides and polyalcohols as β-galactosidase-protecting compounds were characterized at three different concentrations. Disaccharides were generally more effective than polyalcohols, which appeared to be caused in some cases by their lower crystallization tendency. Four disaccharides (cellobiose, melibiose, sucrose and trehalose) were selected for further secondary structure stabilization studies, which corroborated the results of the activity analysis studies that the protein-protecting efficacies of trehalose and melibiose differed from those of sucrose and cellobiose.Secondly, the water plasticization and crystallization properties of the four disaccharides were studied after freeze drying by storing them at different relative humidity atmospheres. Cellobiose and sucrose were shown to crystallize faster and at lower relative humidity than trehalose and melibiose. The water plasticization properties of sucrose were shown to differ from the other disaccharides. Melibiose was most stable of the four compounds in amorphous state, which may have partly been caused by its slower molecular relaxation rate compared to trehalose.Thirdly, the effect of freeze drying parameters on polyclonal IgG stability was studied in order to find out how much the stability of such antibodies could be affected by processing conditions in formulations containing trehalose. While varying the primary drying pressure and secondary drying heating rate did not directly affect protein stability, increased aggregation did occur at lower primary drying pressures when sodium phosphate buffer was used in the formulation. This may have been caused by buffer freeze-concentration, which may have been more significant when the sample temperature was lowered below the glass transition temperature of the freeze-concentrated phase. Sodium chloride was also shown to reduce the Z-average diameter of the rehydrated polyclonal IgG lyophilizates.Finally, the efficacies of trehalose and melibiose in protecting a monoclonal antibody (rituximab) from degradation during freeze drying and storage at different relative humidity atmospheres were compared by storing the lyophilizates up to 3 months. Melibiose was generally more effective than trehalose in inhibiting secondary structure alterations, fragmentation and aggregation during drying and storage. Storage at dry conditions (RH 5{\%}) was shown to be more harmful for rituximab in the lyophilized formulations than storage at more humid conditions (RH 11-23{\%}), which may have been caused by the susceptibility of the protein to drying-induced degradation.",
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author = "Petteri Heljo",
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language = "English",
isbn = "978-952-10-8616-8",
series = "Dissertationes Biocentri Viikki Universitatis Helsingiensis",
publisher = "University of Helsinki",
number = "4",
address = "Finland",

}

Comparison of disaccharides and polyalcohols as stabilizers in freeze-dried protein formulations. / Heljo, Petteri.

Helsinki : University of Helsinki, 2013. 64 p.

Research output: ThesisDoctoral ThesisCollection of Articles

TY - THES

T1 - Comparison of disaccharides and polyalcohols as stabilizers in freeze-dried protein formulations

AU - Heljo, Petteri

PY - 2013

Y1 - 2013

N2 - Protein-structured pharmaceuticals have become very important for treating several chronic and acute illnesses, but their commercialization is sometimes impeded by their poor stability in aqueous solutions. In some cases, protein stability may be better in dry than in aqueous formulations, and freeze drying (lyophilization) is the most commonly used method for producing such products. However, excipients are needed to keep the proteins from degrading during processing and storage, and the current selection of stabilizing compounds accepted by the regulatory authorities is limited. The aim of this thesis was to compare the stabilizing efficacies of different disaccharides and polyalcohols in protein formulations.Firstly, the efficacies of 11 disaccharides and polyalcohols as β-galactosidase-protecting compounds were characterized at three different concentrations. Disaccharides were generally more effective than polyalcohols, which appeared to be caused in some cases by their lower crystallization tendency. Four disaccharides (cellobiose, melibiose, sucrose and trehalose) were selected for further secondary structure stabilization studies, which corroborated the results of the activity analysis studies that the protein-protecting efficacies of trehalose and melibiose differed from those of sucrose and cellobiose.Secondly, the water plasticization and crystallization properties of the four disaccharides were studied after freeze drying by storing them at different relative humidity atmospheres. Cellobiose and sucrose were shown to crystallize faster and at lower relative humidity than trehalose and melibiose. The water plasticization properties of sucrose were shown to differ from the other disaccharides. Melibiose was most stable of the four compounds in amorphous state, which may have partly been caused by its slower molecular relaxation rate compared to trehalose.Thirdly, the effect of freeze drying parameters on polyclonal IgG stability was studied in order to find out how much the stability of such antibodies could be affected by processing conditions in formulations containing trehalose. While varying the primary drying pressure and secondary drying heating rate did not directly affect protein stability, increased aggregation did occur at lower primary drying pressures when sodium phosphate buffer was used in the formulation. This may have been caused by buffer freeze-concentration, which may have been more significant when the sample temperature was lowered below the glass transition temperature of the freeze-concentrated phase. Sodium chloride was also shown to reduce the Z-average diameter of the rehydrated polyclonal IgG lyophilizates.Finally, the efficacies of trehalose and melibiose in protecting a monoclonal antibody (rituximab) from degradation during freeze drying and storage at different relative humidity atmospheres were compared by storing the lyophilizates up to 3 months. Melibiose was generally more effective than trehalose in inhibiting secondary structure alterations, fragmentation and aggregation during drying and storage. Storage at dry conditions (RH 5%) was shown to be more harmful for rituximab in the lyophilized formulations than storage at more humid conditions (RH 11-23%), which may have been caused by the susceptibility of the protein to drying-induced degradation.

AB - Protein-structured pharmaceuticals have become very important for treating several chronic and acute illnesses, but their commercialization is sometimes impeded by their poor stability in aqueous solutions. In some cases, protein stability may be better in dry than in aqueous formulations, and freeze drying (lyophilization) is the most commonly used method for producing such products. However, excipients are needed to keep the proteins from degrading during processing and storage, and the current selection of stabilizing compounds accepted by the regulatory authorities is limited. The aim of this thesis was to compare the stabilizing efficacies of different disaccharides and polyalcohols in protein formulations.Firstly, the efficacies of 11 disaccharides and polyalcohols as β-galactosidase-protecting compounds were characterized at three different concentrations. Disaccharides were generally more effective than polyalcohols, which appeared to be caused in some cases by their lower crystallization tendency. Four disaccharides (cellobiose, melibiose, sucrose and trehalose) were selected for further secondary structure stabilization studies, which corroborated the results of the activity analysis studies that the protein-protecting efficacies of trehalose and melibiose differed from those of sucrose and cellobiose.Secondly, the water plasticization and crystallization properties of the four disaccharides were studied after freeze drying by storing them at different relative humidity atmospheres. Cellobiose and sucrose were shown to crystallize faster and at lower relative humidity than trehalose and melibiose. The water plasticization properties of sucrose were shown to differ from the other disaccharides. Melibiose was most stable of the four compounds in amorphous state, which may have partly been caused by its slower molecular relaxation rate compared to trehalose.Thirdly, the effect of freeze drying parameters on polyclonal IgG stability was studied in order to find out how much the stability of such antibodies could be affected by processing conditions in formulations containing trehalose. While varying the primary drying pressure and secondary drying heating rate did not directly affect protein stability, increased aggregation did occur at lower primary drying pressures when sodium phosphate buffer was used in the formulation. This may have been caused by buffer freeze-concentration, which may have been more significant when the sample temperature was lowered below the glass transition temperature of the freeze-concentrated phase. Sodium chloride was also shown to reduce the Z-average diameter of the rehydrated polyclonal IgG lyophilizates.Finally, the efficacies of trehalose and melibiose in protecting a monoclonal antibody (rituximab) from degradation during freeze drying and storage at different relative humidity atmospheres were compared by storing the lyophilizates up to 3 months. Melibiose was generally more effective than trehalose in inhibiting secondary structure alterations, fragmentation and aggregation during drying and storage. Storage at dry conditions (RH 5%) was shown to be more harmful for rituximab in the lyophilized formulations than storage at more humid conditions (RH 11-23%), which may have been caused by the susceptibility of the protein to drying-induced degradation.

KW - 317 Pharmacy

M3 - Doctoral Thesis

SN - 978-952-10-8616-8

T3 - Dissertationes Biocentri Viikki Universitatis Helsingiensis

PB - University of Helsinki

CY - Helsinki

ER -

Heljo P. Comparison of disaccharides and polyalcohols as stabilizers in freeze-dried protein formulations. Helsinki: University of Helsinki, 2013. 64 p. (Dissertationes Biocentri Viikki Universitatis Helsingiensis; 4).