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Optimization of an engineered IgG binding protein domain with calcium dependent behavior for mild antibody purification

Scheffel, Julia LU (2017) KBK820 20171
Pure and Applied Biochemistry
Abstract
Antibodies have become immensely important reagents within biomolecular research and analysis as well as in medicine. Monoclonal antibodies are the largest class of biological drugs and with an increasing number of therapeutic antibodies approved for clinical use the need for efficient
purification strategies is vast. Today, most antibodies are purified using affinity chromatography based on Protein A, an extensively researched protein. However, a major drawback of Protein A is the need for a low pH (2-3) to elute the antibodies from the purification column. Since acidic
pH poses the risk of aggregation and even loss of function of the antibody, these harsh elution conditions have been avoided by the development of a Protein A based IgG... (More)
Antibodies have become immensely important reagents within biomolecular research and analysis as well as in medicine. Monoclonal antibodies are the largest class of biological drugs and with an increasing number of therapeutic antibodies approved for clinical use the need for efficient
purification strategies is vast. Today, most antibodies are purified using affinity chromatography based on Protein A, an extensively researched protein. However, a major drawback of Protein A is the need for a low pH (2-3) to elute the antibodies from the purification column. Since acidic
pH poses the risk of aggregation and even loss of function of the antibody, these harsh elution conditions have been avoided by the development of a Protein A based IgG binding protein domain Zmat8 with calcium dependent behavior. The antibodies can instead be purified by EDTA at pH
5.5, and in this master thesis project the protein domain Zmat8 is optimized by the development of multimeric variants. The multimeric variants were designed and cloned followed by production and purification of the proteins Zmat8MonoCys, Zmat8DiCys, Zmat8TriCys and Zmat8TetCys.
They were immobilized onto a cysteine reactive matrix and tested as affinity chromatography ligands in an IgG-purification set up. The aim was to find a ligand with a higher binding capacity than the monomer that can purify larger amounts of IgG. Based on the purification tests, the amount of eluted IgG appears to increase with the length of the protein ligand and subside after
Zmat8TriCys. Zmat8TriCys and Zmat8TetCys appear to cause the most efficient antibody purification with slightly more IgG eluted by Zmat8TriCys. Consequently, this master thesis project has optimized the novel protein domain Zmat8 by enabling more efficient antibody purification at mild elution conditions with significantly reduced risks of aggregation or loss of
function compared to traditional Protein A affinity chromatography. (Less)
Popular Abstract
Proteins that are built by several identical subunits are shown to be more efficient tools to isolate antibodies from a mixture of proteins compared to a protein with one single subunit. That is likely because the proteins of several subunits are large enough to catch and fish out more antibodies out of the mixture.
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author
Scheffel, Julia LU
supervisor
organization
course
KBK820 20171
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Calcium dependent, Zmat8, Protein A, Antibodies, Purification, Multimeric variants, applied biochemistry, tillämpad biokemi
language
English
id
8905976
date added to LUP
2017-09-19 15:26:55
date last changed
2017-09-19 15:26:55
@misc{8905976,
  abstract     = {Antibodies have become immensely important reagents within biomolecular research and analysis as well as in medicine. Monoclonal antibodies are the largest class of biological drugs and with an increasing number of therapeutic antibodies approved for clinical use the need for efficient
purification strategies is vast. Today, most antibodies are purified using affinity chromatography based on Protein A, an extensively researched protein. However, a major drawback of Protein A is the need for a low pH (2-3) to elute the antibodies from the purification column. Since acidic
pH poses the risk of aggregation and even loss of function of the antibody, these harsh elution conditions have been avoided by the development of a Protein A based IgG binding protein domain Zmat8 with calcium dependent behavior. The antibodies can instead be purified by EDTA at pH
5.5, and in this master thesis project the protein domain Zmat8 is optimized by the development of multimeric variants. The multimeric variants were designed and cloned followed by production and purification of the proteins Zmat8MonoCys, Zmat8DiCys, Zmat8TriCys and Zmat8TetCys.
They were immobilized onto a cysteine reactive matrix and tested as affinity chromatography ligands in an IgG-purification set up. The aim was to find a ligand with a higher binding capacity than the monomer that can purify larger amounts of IgG. Based on the purification tests, the amount of eluted IgG appears to increase with the length of the protein ligand and subside after
Zmat8TriCys. Zmat8TriCys and Zmat8TetCys appear to cause the most efficient antibody purification with slightly more IgG eluted by Zmat8TriCys. Consequently, this master thesis project has optimized the novel protein domain Zmat8 by enabling more efficient antibody purification at mild elution conditions with significantly reduced risks of aggregation or loss of
function compared to traditional Protein A affinity chromatography.},
  author       = {Scheffel, Julia},
  keyword      = {Calcium dependent,Zmat8,Protein A,Antibodies,Purification,Multimeric variants,applied biochemistry,tillämpad biokemi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Optimization of an engineered IgG binding protein domain with calcium dependent behavior for mild antibody purification},
  year         = {2017},
}