Protein Purification Practical Report

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Protein Purification Practical Report

Student Name : Noel Murphy

Date : 28 February 2015

UF/DF Practical Question Report

Ultrafiltration (UF) and Diafiltration (DF) steps are carried out prior to every chromatography step and post the final polishing chromatography step. These UF and DF steps are vitally important, the UF step concentrates the protein product into a smaller volume of buffer, while the DF step alters the conditions the protein product is sitting in i.e. Buffer exchange. These steps are required as they concentrate the protein product in smaller volumes of buffer allowing smaller equipment to be used, reduced time and resources and also ensures the protein product is in the correct buffer for the next step in
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This mode of filtration was developed as a method of reducing fouling on the filter membrane and thus improves filtration efficiency. This process uses filter cassettes and the direction of the fluid stream is parallel to the filter under an applied pressure. These filter cassettes are expensive to purchase, but are reusable once the filter cassettes have been cleaned to remove all traces of residual protein, foulants and other contaminants from the previous UF/ DF run. Prior to running a UF/ DF cycle for the first time, clean water is flushed through the filter membrane to ensure proper performance of the membranes. This test is called a Normalised Water Permeability (NWP) test.

Normalised Water Permeability is a measure of the flow rate of clean water through the filter membranes under the same transmembrane pressure that will be used during the processing steps. This test is performed as it tests the permeability of water through a membrane, it calculates the flow rate of the water through the membrane, it tests the effectiveness of the cleaning process and also provides a trend of the value of repeated cycles which in hand can be used to gauge the expected lifecycle of a filter and thus set a limit on the maximum number of runs that can be performed on one set of filter cassettes.
To check the efficiency of a cleaning run post a UF/DF step, a Membrane Recovery
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This system consists of two filters in sequence, a depth filter followed by a sterile grade 0.22µm filter, utilising this sterile grade filter doesn’t mean sterility is claimed, but it is used to further improve the clarification process and help reduce the bioburden count. The depth filter uses the filter medium to retain particles throughout the filter medium, the larger particles retained do not cause filter membrane blinding and the smaller particles are retained deeper in the membrane, thus allowing for better filtration efficiency and clarification of the protein culture

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