The Novel Exploration And Development of Single-use, Single-pass Continuous Downstream Bioprocessing Technologies Using Hollow Fiber Membranes
Open Access
- Author:
- Yehl, Christopher
- Graduate Program:
- Chemical Engineering (PHD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 13, 2021
- Committee Members:
- Andrew Zydney, Chair & Dissertation Advisor
William Hancock, Outside Unit & Field Member
Esther Gomez, Major Field Member
Stephanie Velegol, Major Field Member
Seong Han Kim, Professor in Charge/Director of Graduate Studies - Keywords:
- Biotherapeutics
Downstream Processing
Hollow Fibers
Continuous Processing
Single-use
Single-Pass
Ultrafiltration
Diafiltration
Countercurrent Dialysis
Monoclonal Antibodies
High Performance Countercurrent Membrane Purification
Hollow Fiber Characterization
Hollow Fiber Modification - Abstract:
- There are growing interests and pressures in industry to shift the manufacturing of biotherapeutics from a sequence of batch operation to a fully continuous process. With technological advancements in upstream processing providing higher product titers, the strain and apparent bottlenecks on traditionally used downstream processes has never been greater. Thus, the implementation of continuous bioprocessing is now an obtainable goal in the foreseeable future. Although there have been great advances on the upstream side of bioprocessing, the advancements on the downstream side have progressed more slowly. Few candidate systems and technologies have been developed, explored, and marketed as replacements to the current status quo of batch processing. Even when a likely candidate emerges, other issues such as efficiency, scalability, cost, reusability, and cleanability, can significantly reduce the viability of the technology. The overall objective of this thesis is to explore new membrane-based processes, modules, and techniques that can be used to advance continuous bioprocessing technology for the manufacture of biotherapeutics and to address some of the challenges in replacing current technologies. The specific aims include 1) evaluating continuous processing replacement technologies for buffer exchange via diafiltration, 2) evaluate hollow fiber technologies for continuous protein concentration by ultrafiltration, 3) develop new characterization protocols appropriate for these hollow fiber membrane technologies, 4) explore a novel countercurrent membrane contacting process for protein purification and isolation, and 5) develop theoretical frameworks that can help optimize and evaluate the proposed continuous processes outlined above. Most of the experiments discussed in this thesis were performed using hollow fiber hemodialysis membranes cast from a blend of polyethersulfone and polyvinylpyrrolidone. These hemodialyzers, which to date have been exclusively used in the medical industry, are an ideal candidate for continuous bioprocessing due to their low-cost, biocompatibility, large effective surface area, high packing density, and easy scalability. This thesis will clearly show that these hollow fiber dialyzers can not only achieve desirable industry performance targets but can in many cases significantly out-perform existing technologies used in both ultrafiltration and diafiltration. The results from Chapters 1-3, as it relates to replacing diafiltration with countercurrent dialysis and replacing traditional batch ultrafiltration with single pass tangential flow filtration, show that the target 1000-fold buffer exchange can easily be achieved using countercurrent dialysis (CCD), and that this approach consumes 6x less buffer than tradition batch diafiltration. The results will show that only 2-4 modules will be needed operating in series to perform buffer exchange for a 2000 L perfusion bioreactor when placed inline after an ultrafiltration step (depending on the UF concentration). Chapter 3 will show single pass tangential flow filtration experiments that will provide more than 10-fold concentration of immunoglobulin G (IgG) in a single-pass with exit concentrations greater than 200 g/L, along with stable operation during a continuous run of over 120 hours. This chapter will show that single dialyzer will be able to process 1.4 kg of IgG per day with a total cost of less than $0.004 per g of IgG (based on the module cost). Results will also show that running two modules in series will be capable of continuous ultrafiltration for a 2000 L perfusion bioreactor. Chapter 4 will provide new techniques and valuable insights into more effective characterization protocols for evaluating the dextran retention curves and effective pore size of large molecular weight cut-off hollow fiber membranes using a combination of experimental measurements and theoretical modeling. The results will provide fundamental insights into the factors controlling dextran retention and the calculated molecular weight cutoff (MWCO), including both concentration polarization and backfiltration. The results in chapters 5-7 will examine the development and application of a new, patent pending, process technology that we will refer to as High Performance Countercurrent Membrane Purification (HPCMP) that could significantly reduce/minimize the costs of the initial product capture and purification steps while providing the foundation for a truly continuous downstream process. Chapter 5 will present experiments using commercially available hemodialysis membranes to explore the separation capabilities of HPCMP. The HPCMP system will then separate bovine serum albumin (BSA) and myoglobin (Mb), to achieve greater than 98% yield of both proteins with purification factors greater than 100-fold. Stable operation was achieved for 96 hours without any evidence of membrane fouling due to the absence of any significant pressure-driven flow. Chapter 6 will present a set of optimization equations and diagrams that will describe the tradeoff between the yield and purification factor in HPCMP processes in terms of two parametric variables: the diffusive membrane selectivity and the ratio of the draw to bulk solution flow rates. Model calculations will also be used to quantify the greater selectivity of diffusive transport compared to traditional pressure-driven membrane separations for membranes with the same pore size distribution. The results will provide a framework that can be used for the design and optimization of HPCMP processes for highly selective protein separations. Chapter 7 will further explore the feasibility of using HPCMP for high resolution separations in the preparation of biopharmaceuticals and natural protein products. The chapter will outline a modification technique for shifting the pore size distribution in commercially available hemodialysis membranes to enable the separation of larger species. The tailored HPCMP system will separate BSA and IgG, achieving greater than 97% yield of both proteins with purification factors and selectivity as great as 50-fold. Stable operation will show 96 hours of continuous operation without any evidence of membrane fouling due to the absence of any significant pressure-driven flow.