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Title: Microscale methods to establish scalable operations for protein impurity removal prior to packed bed steps
Author: Hanif, R.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2013
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The purification of monoclonal antibodies and Fab’ antibody fragments are of central importance to the pharmaceutical industry. In 2008, 29 new therapies based on such molecules were approved for the US market. Traditionally, a multistep process achieves purification with the majority of steps being packed bed chromatography. Chromatography is the major contributor to the unit operation costs in terms of initial capital expenditure for packing and recurrent replacement costs. When considering the demand for biopharmaceuticals, it becomes necessary to consider alternative process strategies to improve the economics of purification of such proteins. To address this issue, this thesis investigates precipitation to selectively isolate Fab’ or remove protein impurities to assist the initial purification process. The hypothesis tested was that the combination of two or more precipitating agents will alter the solubility profile of Fab’ or protein impurities through synergistic multimodal effects. This principle was investigated through combinations of polyethylene glycol (PEG) with ammonium sulphate, sodium citrate and sodium chloride at different ratios in a novel multimodal approach. A high throughput system utilising automated robotic handling was developed in microwells at 1 mL scale per well to enable the rapid screening of a large number of variables in parallel using a Design of Experiments (DoE) approach to statistically design studies in a two stage process, based on Quality by Design principles. In the first stage, Fab’ precipitation using PEG was investigated using a screening study in the form of a full two level factorial DoE to investigate a large design space. This was followed by a second more focused central composite face centred DoE to find optimal experimental conditions to deliver a high Fab’ yield and purification factor in the range investigated. A design space comprised of the responses percentage Fab’ yield and purification factor was created to give a robust region where Fab’ yield was ≥ 90% with a maximum purification factor of 1.7. A normal operating range (NOR) was defined within this design space for operational simplicity when working at process scale. A confirmatory run was performed within the NOR with PEG 12000 15% w/v pH 7.4, which delivered a Fab’ yield and purification factor of 93% and 1.5 respectively. In the second stage, optimum conditions from the first study were used in a central composite face centred DoE incorporating multimodal conditions combining PEG with three salts from the Hofmeister series namely, ammonium sulphate, sodium citrate and sodium chloride. It was found that 90% Fab’ yield with a purification factor of 1.9 was achievable with PEG 12000 15% w/v/0.30 M sodium citrate/0.15 M ammonium sulphate pH 7.4. This was an improvement of 26% relative to the use of 15% w/v PEG 12000 pH 7.4 in single mode. However an alternative precipitation strategy to precipitate ~20% of protein impurities whilst Fab’ remained soluble using PEG 12000 6.25% w/v/0.4 M sodium citrate pH 7.4 was proposed instead. The advantage of this approach at process scale is the potential ease of processing due to removal of a solubilisation step and the significantly reduced viscosity of the precipitating agent relative to that of high concentrations of PEG. It was shown that this system could mimic process scale, which was verified at laboratory scale (50 mL stirred tank reactor (STR)) and pilot process scale (5 L STR). A process run through was performed using a 1 mL SP Sepharose Hi Trap pre packed bed column (GE Healthcare, Uppsala, Sweden) to capture Fab’ from homogenate (control), multimodal (PEG 12000 6.25% w/v/0.4 M sodium citrate pH 7.4) and single mode (PEG 12000 15% w/v pH 7.4) feedstreams. The final process purification factor for the three feedstreams were 2.5, 4.4 and 3.5 respectively. The use of multimodal precipitated impurities prior to a packed bed step had improved process performance by a purification factor of 1.9. This underlines the importance of assessing the interaction of individual processing steps, and the implementation of appropriate scale down models as a means of achieving process parameter ranging understanding. The impact of which has the further potential to improve the longevity of chromatography resins and reducing overall downstream purification cost.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available