An evaluation of bioprocess development approaches for the precipitation-based purification of IgG monoclonal antibodies.
Doctoral thesis, UCL (University College London).
This thesis investigates bioprocess development approaches for the precipitation-based purification of IgG monoclonal antibodies (mAb) from clarified cell culture supernatant. A high throughput platform using microgram quantities of IgG was developed for the rapid identification and selection of suitable conditions for precipitation of IgG from a complex feedstock. The most effective conditions were then assessed in an ultra scale-down (USD) device run with 60 mL of material in order to characterise the precipitates in terms of their particle size distribution (PSD), morphology, strength and density with the aim of predicting likely precipitation and centrifugation separation performance upon scale-up. The influence of the physiochemical environment on precipitation was investigated and the effectiveness of the USD device in predicting laboratory scale and pilot scale performance was also evaluated. A high throughput micro-scale approach conducted in a 96 microwell plate format was developed for scouting favourable precipitation conditions. Optical measurements were used to locate suitable conditions. Turbidity was measured by absorbance at 600 nm as a rapid measure of the solids content and to monitor the progress of precipitation. Coupled with high throughput analytical techniques including Protein A HPLC and capillary SDS electrophoresis, the strategy for rapid process screening was defined. The capacity of this system to deliver rapid process understanding was then illustrated by applying it to the full factorial investigation of Polyethylene Glycol (PEG) precipitation for an IgG mAb as a function of antibody concentration, precipitant concentration and pH. Results showed that PEG concentrations required for maximum yield and purity were dependent on the IgG concentration. A window of operation was defined for all mAb concentrations tested where PEG concentrations of 12 to 20 %w/v, pH 8.0 gave desirable levels of yield and purity. The effect of reactor conditions on the IgG precipitate characteristics generated from batch precipitations at USD scale showed a marked dependence of PSD on mixing, IgG concentration, PEG concentration, and PEG addition rate. The results also showed that particle strength and density were a factor of aging time, PEG concentration and mixing intensity. Data on scale-up from USD to lab (330 mL) and pilot (7 to 8 L) scales indicated that the trends and parameters identified at USD scale were important at large scale. Scale-up based on the local energy dissipation rate of the impeller region and the ratio of circulation time to feed addition time was shown to allow accurate prediction of large scale performance from USD scale. A USD rotating shear device providing feed material to a laboratory batch centrifuge allowed successful prediction of pilot plant scale clarification performance using the equivalent settling area theory. Expectatedly, preliminary analysis of the bulk re-solubilised precipitates following the precipitation step showed that the level of impurity clearance achieved did not match the performance of traditional Protein A affinity chromatography. However, integrating the precipitation step into a generic antibody purification platform and substituting the Protein A chromatography capture step with a precipitation step demonstrated the potential of precipitation albeit purity and yield of the final purified product was lower than the process incorporating a Protein A capture step. Nevertheless, this work indicated that further development activities would be required in order to use precipitation in the biopharmaceutical manufacture of mAbs. The use of precipitation for antibody recovery could potentially reduce costs associated with downstream operations, increase plant throughput and manage large intermediate process volumes associated with high titre high volume mAb production processes. Furthermore, such a development and scale-up approach could reduce the timelines for bioprocess design and development to facilitate faster drug to clinic initiatives. In conclusion, this study has demonstrated that micro-scale and ultra scale-down evaluation methods can be used successfully to predict precipitation conditions for use at laboratory and pilot scales. Rapid development of precipitation conditions can allow this unit operation to be used for a range of recombinant proteins (including mAbs) with associated reductions in cost of manufacturing.
|Title:||An evaluation of bioprocess development approaches for the precipitation-based purification of IgG monoclonal antibodies|
|Additional information:||Permission for digitisation not received|
|UCL classification:||UCL > School of BEAMS > Faculty of Engineering Science > Biochemical Engineering|
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