Analysis of tradeoffs between purification factor and yield for high-performance countercurrent membrane purification for protein separations.

High-performance countercurrent membrane purification (HPCMP) has recently been presented as a new approach for protein separations, exploiting differences in diffusive transport across a semipermeable membrane to achieve high selectivity for protein separations. This study presents a set of design equations and diagrams that 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. Conditions are identified that provide the high yields and purification factors of interest in bioprocessing. In addition, hydrodynamic models for solute transport were used to evaluate the selectivity as a function of the membrane pore size distribution for purely size-based separations. Model calculations demonstrate that diffusive transport provides significantly greater selectivity than traditional pressure-driven membrane separations for the same pore size distribution due to differences in hindered transport rates for diffusion and convection. These results provide a framework that can be used for the development of HPCMP processes for highly selective protein separations.

Multistage continuous countercurrent diafiltration for formulation of monoclonal antibodies.

There is growing interest in the development of fully integrated and continuous biomanufacturing processes for the production of monoclonal antibody products. A recent study has demonstrated the feasibility of using a two-stage countercurrent diafiltration (DF) process for continuous product formulation, but this system did not provide sufficient levels of buffer exchange for most applications. The objective of this study was to design and test a three-stage countercurrent DF system that could achieve at least 99.9% buffer exchange over 24 hr of continuous operation. Experimental data were obtained using concentrated solutions of human immunoglobulin G as a model protein, with the extent of vitamin B12 removal used to track the extent of DF. Pall Cadence™ inline concentrators with Delta 30 kD regenerated cellulose membranes were used in the three stages to achieve high conversion in a single pass. The three-stage system showed stable operation with >99.9% vitamin B12 removal and a minimal increase in pressure over the full 24 hr. Modules were effectively cleaned using sodium hydroxide, with nearly complete recovery of water permeability. A simple economic analysis was presented that accounts for the trade-offs between quantity of buffer used and membrane costs for this type of countercurrent staged DF process. The results provide important insights to the design and operation of a continuous process for antibody formulation.

Hollow fiber countercurrent dialysis for continuous buffer exchange of high-value biotherapeutics.

Buffer exchange, desalting, and formulation of high-value biotherapeutics are currently performed using batch diafiltration (DF); however, this type of tangential flow filtration process may be difficult to implement as part of a fully continuous biomanufacturing process. The objective of this study was to explore the potential of using countercurrent dialysis for continuous protein formulation and buffer exchange. Experiments were performed using concentrated solutions of immunoglobuin G (IgG) with commercially available hollow fiber dialyzers having 1.5 and 1.8 m2 membrane surface area. More than 99.9% buffer exchange was obtained over a range of conditions, as determined from the removal of a model impurity (vitamin B12 ). The dialyzers were able to process more than 0.5 kg of IgG per day in an easily scalable low-cost process. In addition, buffer requirements were less than 0.02 L of buffer per gram IgG, which is several times less than that used in current batch DF processes. These results clearly demonstrate the potential of using low-cost hollow fiber dialyzers for buffer exchange and product formulation in continuous bioprocessing. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2763, 2019.

Tipping the Balance between S-π and O-π Interactions.

A comprehensive experimental survey consisting of 36 molecular balances was conducted to compare 18 pairs of S-π versus O-π interactions over a wide range of structural, geometric, and solvent parameters. A strong linear correlation was observed between the folding energies of the sulfur and oxygen balances across the entire library of balance pairs. The more stable interaction systematically switched from the O-π to S-π interaction. Computational studies of bimolecular PhSCH3-arene and PhOCH3-arene complexes were able to replicate the experimental trends in the molecular balances. The change in preference for the O-π to S-π interaction was due to the interplay of stabilizing (dispersion and solvophobic) and destabilizing (exchange-repulsion) terms arising from the differences in size and polarizability of the oxygen and sulfur atoms.

Anion-enhanced solvophobic effects in organic solvent.

The influence of salts on the solvophobic interactions of two non-polar surfaces in organic solvent was examined using a series of molecular balances. Specific anion effects were observed that followed the Hofmeister series and enhanced the solvophobic effect up to two-fold.

Stabilizing Fluorine-π Interactions.

A series of N-arylimide molecular balances were designed to study and measure fluorine-aromatic (F-π) interactions. Fluorine substituents gave rise to increasingly more stabilizing interactions with more electron-deficient aromatic surfaces. The attractive F-π interaction is electrostatically driven and is stronger than other halogen-π interactions.

Measurement of Solvent OH-π Interactions Using a Molecular Balance.

A molecular torsion balance was designed to study and measure OH-π interactions between protic solvents and aromatic surfaces. These specific solvent-solute interactions were measured via their influence on the folded-unfolded equilibrium of an N-arylimide rotor. Protic solvents displayed systematically weaker solvophobic interactions than aprotic solvents with similar solvent cohesion parameters. This was attributed to the formation of OH-π interactions between the protic solvents and the exposed aromatic surfaces in the unfolded conformer that offset the stronger solvophobic effects for protic solvents.