Novel Surfactant Compatibility with Downstream Protein Bioprocesses.

Downstream processing of antibodies consists of a series of steps aimed at purifying the product and ensuring it is delivered to formulators structurally and functionally intact. The process can be complex and time-consuming, involving multiple filtrations, chromatography, and buffer exchange steps that can interfere with product integrity. This study explores the possibility and benefits of adding N-myristoyl phenylalanine polyether amine diamide (FM1000) as a process aid. FM1000 is a nonionic surfactant that is highly effective at stabilizing proteins against aggregation and particle formation and has been extensively explored as a novel excipient for antibody formulations. In this work, FM1000 is shown to stabilize proteins against pumping-induced aggregation which can occur while transporting them between process units and within certain processes. It is also shown to prevent antibody fouling of multiple polymeric surfaces. Furthermore, FM1000 can be removed after some steps and during buffer exchange in ultrafiltration/diafiltration, if needed. Additionally, FM1000 was compared to polysorbates in studies focusing on surfactant retention on filters and columns. While the different molecular entities of polysorbates elute at different rates, FM1000 flows through purification units as a single molecule and at a faster rate. Overall, this work defines new areas of application for FM1000 within downstream processing and presents it as a versatile process aid, where its addition and removal are tunable depending on the needs of each product.

Impact of wet-dry cycling on the phase behavior and compartmentalization properties of complex coacervates.

Wet-dry cycling on the early Earth is thought to have facilitated production of molecular building blocks of life, but its impact on self-assembly and compartmentalization remains largely unexplored. Here, we investigate dehydration/rehydration of complex coacervates, which are membraneless compartments formed by phase separation of polyelectrolyte solutions. Solution compositions are identified for which tenfold water loss results in maintenance, disappearance, or appearance of coacervate droplets. Systems maintaining coacervates throughout the dehydration process are further evaluated to understand how their compartmentalization properties change with drying. Although added total RNA concentrations increase tenfold, RNA concentration within coacervates remains steady. Exterior RNA concentrations rise, and exchange rates for encapsulated versus free RNAs increase with dehydration. We explain these results in light of the phase diagram, with dehydration-driven ionic strength increase being particularly important in determining coacervate properties. This work shows that wet-dry cycling can alter the phase behavior and protocell-relevant functions of complex coacervates.

Antifouling Ion-Exchange Resins.

Large quantities of organic ion-exchange resins are used worldwide for water decontamination and polishing. Fouling by microorganisms and decomposition products of natural organic matter severely limits the lifetime of these resins. Much research has thus been invested in polymer-based antifouling coatings. In the present study, poly(4-styrenesulfonate) (PSS) and a co-polymer of PSS and a zwitterionic group were used to spontaneously coat commercial Dowex 1X8 anion-exchange resin. UV-visible spectroscopy provided a precise measure of the kinetics and amount of PSS sorbed onto or into resin beads. When challenged with Chlamydomonas reinhardtii algae, uncoated resin was rapidly fouled by algae. Coating the resin with either the homopolymer of PSS or the co-polymer with zwitterion eliminated fouling. Using narrow- and wide-molecular-weight distribution PSS, a cutoff molecular weight of about 240 repeat units was found, above which PSS was unable to diffuse into the resin. Thus, only one monolayer of added PSS was sufficient to confer a highly desirable antifouling property on this resin while consuming less than 0.1% of the exchanger capacity. Radioactive sulfate ions were used to probe the kinetics of (self)exchange, which were virtually unaffected by the PSS coating. This resin treatment is a fast, ultra-low-cost step for potentially enhancing the lifetime of ion exchangers.

Polyelectrolyte complex films influence the formation of polycrystalline micro-structures.

Silica-carbonate biomorphs are inorganic materials composed of thousands of crystalline nanorods that assemble complex morphologies such as helices, vessels, and sheets. We investigate the effect on biomorph crystallization of polyelectrolyte complex films that are prepared using the layer-by-layer deposition technique and post-processed to obtain three stable, chemically distinct films. Biomorph growth on poly(diallyldimethylammonium)-dominated substrates (cationic) shows polycrystalline helical and sheet structures bounded by large witherite prisms. Crystallization on poly(styrenesulfonate)-dominated (anionic) and stoichiometric substrates follows a qualitatively different pathway. We observe islands of radial mineral films that over several days extend at a remarkably constant velocity of 0.48 µm h-1 and eventually mineralize the whole substrate. Our work opens exciting avenues for the use of polyelectrolyte films as tunable substrates for biomimetic crystallization.

Intrinsic Properties of Polyelectrolyte Multilayer Membranes: Erasing the Memory of the Interface.

Polyelectrolyte multilayers (PEMUs) are ultrathin membranes made by alternating adsorption of oppositely charged polyelectrolytes on substrates. Although PEMUs have shown exceptional selectivity for certain ion-filtering applications, they usually contain an excess of one of the polyelectrolytes due to the history- and condition-dependent mode of PEMU assembly. This excess charge provides fixed sites for ion exchange, enhancing the concentration of oppositely charged ions. Thus, the ion-permselective properties of PEMUs cannot be compared unless they are assembled under identical conditions. This work demonstrates the enhanced permeability of PEMUs as-made from poly(diallyldimethylammonium) (PDADMA), and poly(styrene sulfonate) (PSS) to ferricyanide as an example of an anion. Annealing by NaCl followed by pairing of excess PDADMA with additional PSS produces an almost stoichiometric film that better reflects the intrinsic transport properties of PEMUs. This pairing, observed in real time using electrochemical methods, occurs at the PEMU/solution interface under countercurrent transport of PSS from solution and excess PDADMA paired with a counterion, termed PDADMA*, from the PEMU bulk. A quantitative comparison of PSS and PDADMA* diffusion reveals the conditions under which PEMU assembly depends on PSS molecular weight and concentration.

Ion distribution in dry polyelectrolyte multilayers: a neutron reflectometry study.

Ultrathin films of complexed polycation poly(diallyldimethylammonium), PDADMA, and polyanion poly(styrenesulfonate), PSS, were prepared on silicon wafers using the layer-by-layer adsorption technique. When terminated with PDADMA, all films had excess PDADMA, which was balanced by counterions. Neutron reflectivity of these as-made multilayers was compared with measurements on multilayers which had been further processed to ensure 1 : 1 stoichiometry of PDADMA and PSS. The compositions of all films, including polymers and counterions, were determined experimentally rather than by fitting, reducing the number of fit parameters required to model the reflectivity. For each sample, acetate, either protiated, CH3COO-, or deuterated, CD3COO-, served as the counterion. All films were maintained dry under vacuum. Scattering length density profiles were constrained to fit reflectivity data from samples having either counterion. The best fits were obtained with uniform counterion concentrations, even for stoichiometric samples that had been exposed to PDADMA for ca. 5 minutes, showing that surprisingly fast and complete transport of excess cationic charge occurs throughout the multilayer during its construction.

Diffusion of Sites versus Polymers in Polyelectrolyte Complexes and Multilayers.

It has long been assumed that the spontaneous formation of materials such as complexes and multilayers from charged polymers depends on (inter)diffusion of these polyelectrolytes. Here, we separately examine the mass transport of polymer molecules and extrinsic sites-charged polyelectrolyte repeat units balanced by counterions-within thin films of polyelectrolyte complex, PEC, using sensitive isotopic labeling techniques. The apparent diffusion coefficients of these sites within PEC films of poly(diallyldimethylammonium), PDADMA, and poly(styrenesulfonate), PSS, are at least 2 orders of magnitude faster than the diffusion of polyelectrolytes themselves. This is because site diffusion requires only local rearrangements of polyelectrolyte repeat units, placing far fewer kinetic limitations on the assembly of polyelectrolyte complexes in all of their forms. Site diffusion strongly depends on the salt concentration (ionic strength) of the environment, and diffusion of PDADMA sites is faster than that of PSS sites, accounting for the asymmetric nature of multilayer growth. Site diffusion is responsible for multilayer growth in the linear and into the exponential regimes, which explains how PDADMA can mysteriously "pass through" layers of PSS. Using quantitative relationships between site diffusion coefficient and salt concentration, conditions were identified that allowed the diffusion length to always exceed the film thickness, leading to full exponential growth over 3 orders of magnitude thickness. Both site and polymer diffusion were independent of molecular weight, suggesting that ion pairing density is a limiting factor. Polyelectrolyte complexes are examples of a broader class of dynamic bulk polymeric materials that (self-) assemble via the transport of cross-links or defects rather than actual molecules.

Self-Assembled Bilayers on Nanocrystalline Metal Oxides: Exploring the Non-Innocent Nature of the Linking Ions.

Self-assembled bilayers on nanocrystalline metal oxide films are an increasingly popular strategy for modulating electron and energy transfer at dye-semiconductor interfaces. A majority of the work to date has relied on ZrII and ZnIV linking ions to assemble the films. In this report, we demonstrate that several different cations (CdII, CuII, FeII, LaIII, MnII, and SnIV) are not only effective in generating the bilayer assemblies but also have a profound influence on the stability and photophysical properties of the films. Bilayer films with ZrIV ions exhibited the highest photostability on both TiO2 and ZrO2. Despite the metal ions having a minimal influence on the absorption/emission energies and oxidation potentials of the dye, bilayers composed of CuII, FeII, and MnII exhibit significant excited-state quenching. The excited-state quenching decreases the electron injection yield but also, for CuII and MnII bilayers, significantly slows the back electron transfer kinetics.

Water and the Glass Transition Temperature in a Polyelectrolyte Complex.

Hydrated polyelectrolyte complexes, H-PECs, have recently started attracting renewed interest as a class of highly solvated/plasticized blends. H-PECs are observed to undergo a transition in mechanical properties close to room temperature. Whether this is a true glass transition has been questioned recently: the material has an unusually low modulus in the "glassy" state and molecular dynamics simulations have suggested temperature-induced dehydration and water structure changes are responsible for the transition. Using in situ infrared spectroscopic methods on thin films of a widely studied H-PEC we find no definitive evidence for changes in the hydration state of functional groups, the water content, or water structure on passing through Tg for stoichiometric and nonstoichiometric H-PECs. These complexes represent a promising platform for fundamental studies of the glass transition, since the coupling between chains can be modified by "doping" the material with salt, which breaks ion pairing cross-links. The Fox equation was used to estimate Tgs for paired and unpaired oppositely charged repeat units.

Toward ion-free polyelectrolyte multilayers: cyclic salt annealing.

Polyelectrolyte multilayers (PEMUs) are made from various combinations of polyanions and polycations. It is now understood that these ultrathin films of polyelectrolyte complex may also incorporate counterions derived from the solutions from which the PEMU was deposited or exchanged into the film postassembly. If these ions are required to compensate nonstoichiometric ratios of polycation and polyanion they cannot leave the film and exert considerable influence on film properties, such as modulus and permeability. These "extrinsic" charges also complicate fundamental studies on PEMUs. We report a method to remove almost all ionic content from a PEMU made of poly(diallyldimethylammonium chloride), PDADMAC, and poly(styrenesulfonate), PSS. In this method, a high salt concentration plasticizes the multilayer past its glass transition, dispersing all the buried excess PDADMA throughout the film. Exposure to a solution of PSS in a lower salt concentration consumes excess PDADMA near the surface without overcompensating with PSS. The process is repeated in a cyclic fashion, removing >95% of the ions charge present in the as-made PEMU.