An important issue in pharmaceutical industry is to develop processes to purify proteins. Thereare many purification methods that can be used for this purpose, but all methods have differentconstraints. A large interest in affinity chromatography methods can be seen but since theaffinity tag sometimes must be removed, a new self-cleaving affinity chromatography methoddeveloped by Cytiva has the potential of competing with existing chromatography methods. Inthis project two processes, one with conventional purification methods and one with the newself-cleaving affinity chromatography method are developed and compared. A protein calleddesigned ankyrin repeat protein, DARPin, is used as the protein of interest since this proteincan be used as a scaffold protein where a large variety of applications have been investigated. During the project, many different chromatography and other purification methods areevaluated. In the conventional process, after performed experiments, a strong anion exchange chromatography is selected as the first step. The second purification step is chosen to be heat-treatment because of the high melting point of the DARPin. In the last step hydrophobic interaction chromatography is used. This results in a purity of 72-97%, a recovery of 20% and ayield of 0,232 mg DARPin/g cell paste. The development was made in 11 weeks. This can becompared with the second process, where the first step is also strong anion exchangechromatography. The new self-cleaving affinity chromatography was used as a second stepgiving a purity of 89-100%, a recovery of 12% and a yield of 0,231 mg DARPin/g cell paste. Thisdevelopment was made in 5 weeks. The project shows that a process with the new self-cleaving chromatography method gives afinal sample that is purer and that this process can be developed in a much shorter time. Thesame amount of protein can also be obtained even though a lower recovery can be seen.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-501665 |
| Date | January 2023 |
| Creators | Larsson, Sofia |
| Publisher | Uppsala universitet, Biokemi |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | UPTEC X ; 23001 |
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