A capture step with protein A is the most common purification step in the downstream purification process of monoclonal antibodies. It is therefore of great importance to increase the knowledge of the interactions involved in this purification technique. The purpose of this master thesis project was to determine the affinity of protein A domains to IgG subclasses by surface plasmon resonance (SPR). Besides the five homologous IgG-binding protein A domains (E, D, A, B, and C) an engineered domain, similar to domain B and used in the protein A media MabSelect Sure™ (GE Healthcare) was included in the study. The domains were expressed in E.coli, affinity purified and immobilized onto sensor chip surfaces by amine coupling…
Contents
1 INTRODUCTION
2 BACKGROUND
2.1 ANTIBODIES
2.1.1 Structure and classification
2.1.2 Therapeutic antibodies
2.1.3 Production and purification of antibodies
2.2 PROTEIN A
2.3 PROTEIN ANALYSIS
2.3.1 MALDI-TOF
2.3.2 Gel electrophoresis
2.4 CHROMATOGRAPHY
2.4.1 Affinity chromatography
2.4.2 Size exclusion chromatography
2.5 SURFACE PLASMON RESONANCE
2.5.1 Instrumentation
2.5.2 Theoretical background
2.5.3 Sensor Chip
2.5.4 Immobilization
2.6 KINETIC MEASUREMENTS
2.7 AIM OF THE STUDY
3 MATERIALS AND METHODS
3.1 CHEMICALS
3.2 ANTIBODIES
3.3 MATERIAL
3.4 INSTRUMENTATION
3.5 EXPRESSION OF PROTEIN A DOMAINS
3.6 PURIFICATION OF PROTEIN A DOMAINS
3.7 PREPARATION OF IGG-FRAGMENTS
3.8 PURIFICATION OF IGG-FRAGMENTS
3.9 PROTEIN ANALYSIS
3.9.1 Analysis of protein A domains
3.9.2 Analysis of IgG and its fragments
3.10 OPTIMIZATION OF BIACORE EXPERIMENTS
3.10.1 Instrumentation
3.10.2 Running buffer
3.10.3 Preparation of ligands
3.10.4 Preparation of analytes
3.10.5 Reference surfaces
3.10.6 Immobilization approaches
3.10.7 Regeneration scouting
3.10.8 Methods for interaction analyses
3.10.9 Interaction analyses with IgG fragments
3.11 AFFINITY DETERMINATION
3.11.1 Interaction analyses with IgG subclasses
3.11.2 Fitting of data to interaction models
4 RESULTS AND DISCUSSION.
4.1 PRODUCTION OF PROTEIN A DOMAINS
4.2 PRODUCTION OF IGG FRAGMENTS
4.3 PROTEIN ANALYSIS
4.3.1 Analysis of protein A domains
4.3.2 Analysis of IgG
4.4 OPTIMIZATION OF BIACORE EXPERIMENTS
4.4.1 Fitting of data to interaction models
4.4.2 Evaluation of baseline stability
4.4.3 Regeneration scouting
4.5 DETERMINATION OF KD-VALUES
5 CONCLUSIONS
FUTURE STUDIES
ACKNOWLEDGEMENTS
REFERENCES
APPENDIX
Author: Nohldén, Sofia
Source: Linköping University
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