Precipitation-filtration process as a platform for continuous purification of monoclonal antibody therapeutics
Open Access
- Author:
- Minervini, Mirko
- Graduate Program:
- Chemical Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 02, 2024
- Committee Members:
- Robert Rioux, Professor in Charge/Director of Graduate Studies
Andrew Zydney, Chair & Dissertation Advisor
William Hancock, Outside Unit & Field Member
Darrell Velegol, Major Field Member
Esther Gomez, Major Field Member - Keywords:
- Monoclonal antibody
precipitation
microfiltration
membrane separation
downstream - Abstract:
- The significant increase in product titers, coupled with the growing focus on continuous bioprocessing, has renewed interest in using target precipitation as a low‐cost alternative to Protein A chromatography for the primary capture of monoclonal antibody (mAb) products. Following precipitation, soluble impurities can be effectively removed using tangential flow microfiltration. The objective of this thesis was to explore the development and design of a continuous platform purification process using precipitation in tubular flow reactors, induced by addition of ZnCl2 as a cross-linking agent and polyethylene glycol as a volume exclusion agent, coupled with tangential flow microfiltration for dewatering (concentrating) and washing of the precipitated product. One of the major challenges in developing this type of continuous precipitation-based process is ensuring stable operation of the tangential flow microfiltration membranes used for dewatering and washing operations. Continuous (long-time) operation of these membrane modules could be accomplished by maintaining the filtrate flux below the critical or sustainable flux for fouling, which is the maximum value of the flux that causes minimal increase in the transmembrane pressure. The sustainable flux was found to be a function of both the membrane module geometry as well as the properties of the precipitate particles, which are, in turn, influenced by the precipitation conditions. Fiber blockage emerged as a major constraint, particularly in narrow diameter hollow fiber modules, significantly limiting their operational capacity. Additionally, the sustainable flux was found to be highly correlated with the particle packing density measured through centrifugation, which provides a simple approach for rapid screening of the filterability of precipitates generated with different precipitants / concentrations. It was demonstrated for the first time that it is possible to control precipitate densification, and filterability, by addition of several different neutral salts to control the ionic background during the precipitation step, with the best performance achieved using CaCl2. The feasibility and performance of an integrated membrane-based process for the continuous pre-conditioning of clarified cell culture fluid prior to precipitation were also examined. A continuous single pass tangential flow filtration process was demonstrated with 20-fold volume reduction, achieving a final antibody concentration of 56 g/L. A subsequent high performance countercurrent membrane purification process provided more than 50% reduction in host cell proteins, a 30-fold reduction in DNA, and a nearly complete buffer exchange which can provide significant improvements in the efficiency of the subsequent precipitation-filtration and flow- through chromatography processes. These results were used to design and successfully demonstrate the platformability of a continuous precipitation-based capture purification across several different monoclonal antibody products. Final host cell protein and DNA levels were comparable to that achieved using existing processes based on a Protein A affinity chromatography capture step. In addition, the precipitation- filtration process required less buffer per kg of antibody than competitive processes. These results provide important insights for the development of fully integrated (end-to-end) continuous manufacturing processes for high value biotherapeutics exploiting precipitation-filtration for protein capture and purification.