Enantioselective antibody fragments: Dissertation

Research output: ThesisDissertationCollection of Articles

Abstract

Antibodies have a unique ability to bind to a wide variety of different molecules, ranging from large protein antigens to low molecular weight organic molecules. The specificity and affinity of the binding varies between antibodies and can be utilised in many applications. Recombinant antibody fragments have advantages over traditional poly- and monoclonal antibodies in production and immobilisation and in the optimisation of their properties. Enantiomers of a chiral compound provide a challenge for separation methods and analytics due to their similar chemical and physical properties. Antibodies provide a highly specific way to fractionate enantiomers in both preparative and analytical applications. In this work two different antibody-based approaches to separate enantiomers of a chiral drug candidate were developed. Antibody fragments were cloned, produced in bacteria and immobilised on a solid affinity support. Repeated affinity purification of enantiomers was achieved in optimised conditions. In the other approach antibody fragments were immobilised inside the nanotubes of an alumina membrane and the bionanomembrane was used to fractionate enantiomers of a racemic mixture. In addition a sample preparation method, antibody-based solid-phase extraction, was developed in a way that can be applied to high-throughput format. Recoveries were comparable to those reported for non-specific sorbents, but with the advantage of the enantioselectivity. The method was used to extract an enantiomer from a spiked buffer or serum. The preparatory sample treatment protocols usually used for serum, e.g. protein precipitation, were not needed. A homology model of one of the antibody fragments was constructed and used to design site-specific mutations in order to adjust the affinity of the antibody to be suitable for the preparative and analytical approaches developed in this work. One of the mutants, ENA5His Tyr96Val, had appropriate properties both in preparative and analytical applications.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Supervisors/Advisors
  • Söderlund, Hans, Supervisor, External person
  • Teeri, Tuula, Supervisor, External person
Award date8 Oct 2004
Place of PublicationEspoo
Publisher
Print ISBNs951-38-6414-6
Electronic ISBNs951-38-6415-4
Publication statusPublished - 2004
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Immunoglobulin Fragments
Enantiomers
Antibodies
Molecules
Aluminum Oxide
Enantioselectivity
Sorbents
Chemical properties
Nanotubes
Purification
Bacteria
Buffers
Proteins
Physical properties
Molecular weight
Monoclonal Antibodies
Throughput
Membranes
Antigens
Recovery

Keywords

  • antibodies
  • recombinant antibodies
  • antibody fragments
  • enantiomers
  • chiral compounds
  • nanotubes
  • bionanomembranes
  • enantioselectivity
  • haptens
  • affinity chromatography

Cite this

Nevanen, T. (2004). Enantioselective antibody fragments: Dissertation. Espoo: VTT Technical Research Centre of Finland.
Nevanen, Tarja. / Enantioselective antibody fragments : Dissertation. Espoo : VTT Technical Research Centre of Finland, 2004. 96 p.
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author = "Tarja Nevanen",
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publisher = "VTT Technical Research Centre of Finland",
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Nevanen, T 2004, 'Enantioselective antibody fragments: Dissertation', Doctor Degree, University of Helsinki, Espoo.

Enantioselective antibody fragments : Dissertation. / Nevanen, Tarja.

Espoo : VTT Technical Research Centre of Finland, 2004. 96 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Enantioselective antibody fragments

T2 - Dissertation

AU - Nevanen, Tarja

PY - 2004

Y1 - 2004

N2 - Antibodies have a unique ability to bind to a wide variety of different molecules, ranging from large protein antigens to low molecular weight organic molecules. The specificity and affinity of the binding varies between antibodies and can be utilised in many applications. Recombinant antibody fragments have advantages over traditional poly- and monoclonal antibodies in production and immobilisation and in the optimisation of their properties. Enantiomers of a chiral compound provide a challenge for separation methods and analytics due to their similar chemical and physical properties. Antibodies provide a highly specific way to fractionate enantiomers in both preparative and analytical applications. In this work two different antibody-based approaches to separate enantiomers of a chiral drug candidate were developed. Antibody fragments were cloned, produced in bacteria and immobilised on a solid affinity support. Repeated affinity purification of enantiomers was achieved in optimised conditions. In the other approach antibody fragments were immobilised inside the nanotubes of an alumina membrane and the bionanomembrane was used to fractionate enantiomers of a racemic mixture. In addition a sample preparation method, antibody-based solid-phase extraction, was developed in a way that can be applied to high-throughput format. Recoveries were comparable to those reported for non-specific sorbents, but with the advantage of the enantioselectivity. The method was used to extract an enantiomer from a spiked buffer or serum. The preparatory sample treatment protocols usually used for serum, e.g. protein precipitation, were not needed. A homology model of one of the antibody fragments was constructed and used to design site-specific mutations in order to adjust the affinity of the antibody to be suitable for the preparative and analytical approaches developed in this work. One of the mutants, ENA5His Tyr96Val, had appropriate properties both in preparative and analytical applications.

AB - Antibodies have a unique ability to bind to a wide variety of different molecules, ranging from large protein antigens to low molecular weight organic molecules. The specificity and affinity of the binding varies between antibodies and can be utilised in many applications. Recombinant antibody fragments have advantages over traditional poly- and monoclonal antibodies in production and immobilisation and in the optimisation of their properties. Enantiomers of a chiral compound provide a challenge for separation methods and analytics due to their similar chemical and physical properties. Antibodies provide a highly specific way to fractionate enantiomers in both preparative and analytical applications. In this work two different antibody-based approaches to separate enantiomers of a chiral drug candidate were developed. Antibody fragments were cloned, produced in bacteria and immobilised on a solid affinity support. Repeated affinity purification of enantiomers was achieved in optimised conditions. In the other approach antibody fragments were immobilised inside the nanotubes of an alumina membrane and the bionanomembrane was used to fractionate enantiomers of a racemic mixture. In addition a sample preparation method, antibody-based solid-phase extraction, was developed in a way that can be applied to high-throughput format. Recoveries were comparable to those reported for non-specific sorbents, but with the advantage of the enantioselectivity. The method was used to extract an enantiomer from a spiked buffer or serum. The preparatory sample treatment protocols usually used for serum, e.g. protein precipitation, were not needed. A homology model of one of the antibody fragments was constructed and used to design site-specific mutations in order to adjust the affinity of the antibody to be suitable for the preparative and analytical approaches developed in this work. One of the mutants, ENA5His Tyr96Val, had appropriate properties both in preparative and analytical applications.

KW - antibodies

KW - recombinant antibodies

KW - antibody fragments

KW - enantiomers

KW - chiral compounds

KW - nanotubes

KW - bionanomembranes

KW - enantioselectivity

KW - haptens

KW - affinity chromatography

M3 - Dissertation

SN - 951-38-6414-6

T3 - VTT Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Nevanen T. Enantioselective antibody fragments: Dissertation. Espoo: VTT Technical Research Centre of Finland, 2004. 96 p.