Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin

Christian Fogt Hjorth; Mathias Norrman; Per-Olof Wahlund; Andrew J. Benie; Bent O. Petersen; Christian M Jessen; Thomas Å. Pedersen; Kirsten Vestergaard; Dorte B Steensgaard; Jan Skov Pedersen; Helle Naver; František Hubálek; Christian Poulsen; Daniel Otzen

doi:10.1016/j.xphs.2016.01.003

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin

Christian Fogt Hjorth, Mathias Norrman, Per-Olof Wahlund, Andrew J. Benie, Bent O. Petersen, Christian M Jessen, Thomas Å. Pedersen, Kirsten Vestergaard, Dorte B Steensgaard, Jan Skov Pedersen, Helle Naver, František Hubálek, Christian Poulsen, Daniel Otzen

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Abstract

A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.

Original language	English
Journal	Journal of Pharmaceutical Sciences
Volume	105
Issue	4
Pages (from-to)	1376-1386
Number of pages	11
ISSN	0022-3549
DOIs	https://doi.org/10.1016/j.xphs.2016.01.003
Publication status	Published - 1 Apr 2016

Keywords

SAXS
ThT assay
X-ray crystallography
covalent insulin dimer
fibrillation
high molecular weight products

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10.1016/j.xphs.2016.01.003

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Hjorth, C. F., Norrman, M., Wahlund, P.-O., Benie, A. J., Petersen, B. O., Jessen, C. M., Pedersen, T. Å., Vestergaard, K., Steensgaard, D. B., Pedersen, J. S., Naver, H., Hubálek, F., Poulsen, C., & Otzen, D. (2016). Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin. Journal of Pharmaceutical Sciences, 105(4), 1376-1386. https://doi.org/10.1016/j.xphs.2016.01.003

@article{80d6629b7955444380c20f3d93f84d38,

title = "Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin",

abstract = "A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.",

keywords = "SAXS, ThT assay, X-ray crystallography, covalent insulin dimer, fibrillation, high molecular weight products",

author = "Hjorth, {Christian Fogt} and Mathias Norrman and Per-Olof Wahlund and Benie, {Andrew J.} and Petersen, {Bent O.} and Jessen, {Christian M} and Pedersen, {Thomas {\AA}.} and Kirsten Vestergaard and Steensgaard, {Dorte B} and Pedersen, {Jan Skov} and Helle Naver and Franti{\v s}ek Hub{\'a}lek and Christian Poulsen and Daniel Otzen",

year = "2016",

month = apr,

day = "1",

doi = "10.1016/j.xphs.2016.01.003",

language = "English",

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Hjorth, CF, Norrman, M, Wahlund, P-O, Benie, AJ, Petersen, BO, Jessen, CM, Pedersen, TÅ, Vestergaard, K, Steensgaard, DB, Pedersen, JS, Naver, H, Hubálek, F, Poulsen, C & Otzen, D 2016, 'Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin', Journal of Pharmaceutical Sciences, vol. 105, no. 4, pp. 1376-1386. https://doi.org/10.1016/j.xphs.2016.01.003

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin. / Hjorth, Christian Fogt; Norrman, Mathias; Wahlund, Per-Olof et al.
In: Journal of Pharmaceutical Sciences, Vol. 105, No. 4, 01.04.2016, p. 1376-1386.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin

AU - Hjorth, Christian Fogt

AU - Norrman, Mathias

AU - Wahlund, Per-Olof

AU - Benie, Andrew J.

AU - Petersen, Bent O.

AU - Jessen, Christian M

AU - Pedersen, Thomas Å.

AU - Vestergaard, Kirsten

AU - Steensgaard, Dorte B

AU - Pedersen, Jan Skov

AU - Naver, Helle

AU - Hubálek, František

AU - Poulsen, Christian

AU - Otzen, Daniel

PY - 2016/4/1

Y1 - 2016/4/1

N2 - A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.

AB - A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.

KW - SAXS

KW - ThT assay

KW - X-ray crystallography

KW - covalent insulin dimer

KW - fibrillation

KW - high molecular weight products

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U2 - 10.1016/j.xphs.2016.01.003

DO - 10.1016/j.xphs.2016.01.003

M3 - Journal article

C2 - 26921119

SN - 0022-3549

VL - 105

SP - 1376

EP - 1386

JO - Journal of Pharmaceutical Sciences

JF - Journal of Pharmaceutical Sciences

IS - 4

ER -

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin

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