Influence of glycosylation on the adsorption of Thermomyces lannuginosus lipase to hydrophobic and hydrophilic surfaces

Charlotte Pinholt; Mathias Fanø; Charlotte Wiberg; Susanne Hostrup; Jens Thostrup Bukrinsky; Sven Frøkjær; Willem Norde; Lene Jørgensen

doi:10.1016/j.ejps.2010.03.021

Influence of glycosylation on the adsorption of Thermomyces lannuginosus lipase to hydrophobic and hydrophilic surfaces

Charlotte Pinholt, Mathias Fanø, Charlotte Wiberg, Susanne Hostrup, Jens Thostrup Bukrinsky, Sven Frøkjær, Willem Norde, Lene Jørgensen

17 Citations (Scopus)

Abstract

In the pharmaceutical industry, protein drugs are modified by, for instance, glycosylation in order to obtain protein drugs with improved delivery profiles and/or increased stability. The effect of glycosylation on protein adsorption behaviour is one of the stability aspects that must be evaluated during development of glycosylated protein drug products.We have studied the effect of glycosylation on the adsorption behaviour of Thermomyces lanuginosus lipase to hydrophobic and hydrophilic surfaces using total internal reflection fluorescence, surface plasmon resonance, far-UV circular dichroism and fluorescence. Three glyco-variants were used, namely the mono-glycosylated wildtype T. lanuginosus lipase, a non-glycosylated variant and a penta-glycosylated variant, the latter two containing one and nine amino acid substitutions, respectively. All the glycosylations were N-linked and contained no charged sugar residues.Glycosylation did not affect the adsorption of wildtype T. lanuginosus lipase to the hydrophobic surfaces. The number of molecules adsorbing per unit surface area, the structural changes occurring upon adsorption, and the orientation upon adsorption were found to be unaffected by the varying glycosylation. However, the interaction with a hydrophilic surface was different between the three glyco-variants. The penta-glycosylated T. lanuginosus lipase adsorbed, in contrast to the two other glyco-variants.In conclusion, adsorption of T. lanuginosus lipase to hydrophobic surfaces was not affected by N-linked glycosylation. Only penta-glycosylated T. lanuginosus lipase adsorbed to the hydrophilic surface, apparently due to its increased net charge of +3 caused by amino acid substitutions in the primary sequence.

Original language	English
Journal	European Journal of Pharmaceutical Sciences
Volume	40
Issue number	4
Pages (from-to)	273-281
ISSN	0928-0987
DOIs	https://doi.org/10.1016/j.ejps.2010.03.021
Publication status	Published - Jul 2010

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Former Faculty of Pharmaceutical Sciences

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10.1016/j.ejps.2010.03.021

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title = "Influence of glycosylation on the adsorption of Thermomyces lannuginosus lipase to hydrophobic and hydrophilic surfaces",

abstract = "In the pharmaceutical industry, protein drugs are modified by, for instance, glycosylation in order to obtain protein drugs with improved delivery profiles and/or increased stability. The effect of glycosylation on protein adsorption behaviour is one of the stability aspects that must be evaluated during development of glycosylated protein drug products.We have studied the effect of glycosylation on the adsorption behaviour of Thermomyces lanuginosus lipase to hydrophobic and hydrophilic surfaces using total internal reflection fluorescence, surface plasmon resonance, far-UV circular dichroism and fluorescence. Three glyco-variants were used, namely the mono-glycosylated wildtype T. lanuginosus lipase, a non-glycosylated variant and a penta-glycosylated variant, the latter two containing one and nine amino acid substitutions, respectively. All the glycosylations were N-linked and contained no charged sugar residues.Glycosylation did not affect the adsorption of wildtype T. lanuginosus lipase to the hydrophobic surfaces. The number of molecules adsorbing per unit surface area, the structural changes occurring upon adsorption, and the orientation upon adsorption were found to be unaffected by the varying glycosylation. However, the interaction with a hydrophilic surface was different between the three glyco-variants. The penta-glycosylated T. lanuginosus lipase adsorbed, in contrast to the two other glyco-variants.In conclusion, adsorption of T. lanuginosus lipase to hydrophobic surfaces was not affected by N-linked glycosylation. Only penta-glycosylated T. lanuginosus lipase adsorbed to the hydrophilic surface, apparently due to its increased net charge of +3 caused by amino acid substitutions in the primary sequence.",

keywords = "Former Faculty of Pharmaceutical Sciences",

author = "Charlotte Pinholt and Mathias Fan{\o} and Charlotte Wiberg and Susanne Hostrup and Bukrinsky, {Jens Thostrup} and Sven Fr{\o}kj{\ae}r and Willem Norde and Lene J{\o}rgensen",

year = "2010",

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T1 - Influence of glycosylation on the adsorption of Thermomyces lannuginosus lipase to hydrophobic and hydrophilic surfaces

AU - Pinholt, Charlotte

AU - Fanø, Mathias

AU - Wiberg, Charlotte

AU - Hostrup, Susanne

AU - Bukrinsky, Jens Thostrup

AU - Frøkjær, Sven

AU - Norde, Willem

AU - Jørgensen, Lene

PY - 2010/7

Y1 - 2010/7

N2 - In the pharmaceutical industry, protein drugs are modified by, for instance, glycosylation in order to obtain protein drugs with improved delivery profiles and/or increased stability. The effect of glycosylation on protein adsorption behaviour is one of the stability aspects that must be evaluated during development of glycosylated protein drug products.We have studied the effect of glycosylation on the adsorption behaviour of Thermomyces lanuginosus lipase to hydrophobic and hydrophilic surfaces using total internal reflection fluorescence, surface plasmon resonance, far-UV circular dichroism and fluorescence. Three glyco-variants were used, namely the mono-glycosylated wildtype T. lanuginosus lipase, a non-glycosylated variant and a penta-glycosylated variant, the latter two containing one and nine amino acid substitutions, respectively. All the glycosylations were N-linked and contained no charged sugar residues.Glycosylation did not affect the adsorption of wildtype T. lanuginosus lipase to the hydrophobic surfaces. The number of molecules adsorbing per unit surface area, the structural changes occurring upon adsorption, and the orientation upon adsorption were found to be unaffected by the varying glycosylation. However, the interaction with a hydrophilic surface was different between the three glyco-variants. The penta-glycosylated T. lanuginosus lipase adsorbed, in contrast to the two other glyco-variants.In conclusion, adsorption of T. lanuginosus lipase to hydrophobic surfaces was not affected by N-linked glycosylation. Only penta-glycosylated T. lanuginosus lipase adsorbed to the hydrophilic surface, apparently due to its increased net charge of +3 caused by amino acid substitutions in the primary sequence.

AB - In the pharmaceutical industry, protein drugs are modified by, for instance, glycosylation in order to obtain protein drugs with improved delivery profiles and/or increased stability. The effect of glycosylation on protein adsorption behaviour is one of the stability aspects that must be evaluated during development of glycosylated protein drug products.We have studied the effect of glycosylation on the adsorption behaviour of Thermomyces lanuginosus lipase to hydrophobic and hydrophilic surfaces using total internal reflection fluorescence, surface plasmon resonance, far-UV circular dichroism and fluorescence. Three glyco-variants were used, namely the mono-glycosylated wildtype T. lanuginosus lipase, a non-glycosylated variant and a penta-glycosylated variant, the latter two containing one and nine amino acid substitutions, respectively. All the glycosylations were N-linked and contained no charged sugar residues.Glycosylation did not affect the adsorption of wildtype T. lanuginosus lipase to the hydrophobic surfaces. The number of molecules adsorbing per unit surface area, the structural changes occurring upon adsorption, and the orientation upon adsorption were found to be unaffected by the varying glycosylation. However, the interaction with a hydrophilic surface was different between the three glyco-variants. The penta-glycosylated T. lanuginosus lipase adsorbed, in contrast to the two other glyco-variants.In conclusion, adsorption of T. lanuginosus lipase to hydrophobic surfaces was not affected by N-linked glycosylation. Only penta-glycosylated T. lanuginosus lipase adsorbed to the hydrophilic surface, apparently due to its increased net charge of +3 caused by amino acid substitutions in the primary sequence.

KW - Former Faculty of Pharmaceutical Sciences

U2 - 10.1016/j.ejps.2010.03.021

DO - 10.1016/j.ejps.2010.03.021

M3 - Journal article

SN - 0928-0987

VL - 40

SP - 273

EP - 281

JO - European Journal of Pharmaceutical Sciences

JF - European Journal of Pharmaceutical Sciences

IS - 4

ER -

Influence of glycosylation on the adsorption of Thermomyces lannuginosus lipase to hydrophobic and hydrophilic surfaces

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