Wayne F. Reed’s research while affiliated with Tulane University and other places

Monitoring membrane-mediated dialysis in real time with static and dynamic light scattering revealed distinctive differences, including reversibility/irreversibility, in the effects of ionic strength (NaCl) and the denaturant guanidine-HCl (Gd) on a synthetic polyelectrolyte and several types of biomacromolecules: protein, polysaccharide, and polyampholyte. Dialysis cycles against aqueous NaCl and Gd, and reverse back to the original aqueous solution, were monitored. The behavior of Na-polystyrenesulfonate was reversible and yielded a detailed polymer physics description. The biomacromolecules additionally showed hydrogen-bonding/hydrophobic (HP) interactions. An interpretive model was developed that considers the interplay among polyelectrolyte, polyampholyte, and HP potential energies in determining the different associative, aggregative, and dissociative behaviors. NaCl isolated purely electrostatic effects, whereas Gd combined electrostatic and HP effects. Some macromolecules showed partially reversible behavior, and others were completely irreversible. The dialysis monitoring method should prove useful for investigating fundamental macromolecular and colloid properties and for drug formulation and stability optimization.

While adeno-associated virus is a leading vector for gene therapy, significant gaps remain in understanding AAV degradation and stability. In this work, we study the degradation of an engineered AAV serotype at physiological pH and ionic strength. Viral particles of varying fractions of encapsulated DNA were incubated between 30 and 60 °C, with changes in molecular weight measured by changes in total light scattering intensity at 90° over time. Mostly full vectors demonstrated a rapid decrease in molecular weight corresponding to the release of capsid DNA, followed by slow aggregation. In contrast, empty vectors demonstrated immediate, rapid colloid-type aggregation. Mixtures of full and empty capsids showed a pronounced decrease in initial aggregation that cannot be explained by a linear superposition of empty and full degradation scattering signatures, indicating interactions between capsids and ejected DNA that influenced aggregation mechanisms. This demonstrates key interactions between AAV capsids and their cargo that influence capsid degradation, aggregation, and DNA release mechanisms in a physiological solution.

A key aspect to vaccine efficacy is formulation stability. Biochemical evaluations provide information on optimal compositions or thermal stability but are routinely validated by ex vivo analysis and not efficacy in animal models. Here we assessed formulations identified to improve or reduce stability of the mucosal adjuvant dmLT being investigated in polio and enterotoxigenic E. coli (ETEC) clinical vaccines. We observed biochemical changes to dmLT protein with formulation or thermal stress, including aggregation or subunit dissociation or alternatively resistance against these changes with specific buffer compositions. However, upon injection or mucosal vaccination with ETEC fimbriae adhesin proteins or inactivated polio virus, experimental findings indicated immunization route and co-administered antigen impacted vaccine immunogenicity more so than dmLT formulation stability (or instability). These results indicate the importance of both biochemical and vaccine-derived immunity assessment in formulation optimization. In addition, these studies have implications for use of dmLT in clinical settings and for delivery in resource poor settings.

It is known that oxygen (O2) stops radical polymerization (RP). Here, it was found that the reaction turn-off occurs abruptly at a threshold concentration of O2, [O2]t, for both free RP and reversible addition-fragmentation chain-transfer polymerization (RAFT). In some reactions, there was a spontaneous re-start of conversion. Three cases were investigated: RP of (i) acrylamide (Am) and (ii) sodium styrene sulfonate (SS) and (iii) Am RAFT polymerization. A controlled flow of O2 into the reactor was employed. An abrupt turn-off was observed in all cases, where polymerization stops sharply at [O2]t and remains stopped when [O2] > [O2]t. In (i), Am acts as a catalytic radical-transfer agent during conversion plateau, eliminating excess [O2], and polymerization spontaneously resumes at [O2]t. In no reaction, the initiator alone was capable of eliminating O2. N2 purge was needed to re-start reactions (ii) and (iii). For (i) and (ii), while [O2] < [O2]t, O2 acts a chain termination agent, reducing the molecular weight (Mw) and reduced viscosity (RV). O2 acts as an inhibitor for [O2] > [O2]t in all cases. The radical-transfer rates from Am* and SS* to O2 are >10,000× higher than the initial chain propagation step rates for Am and SS, which causes [O2]t at very low [O2].

Monitoring of free and controlled radical homo and copolymerization using automatic continuous online monitoring of polymerization reactions (ACOMP) has been successfully carried out for the past 20 years. An ultraviolet (UV) detector, often combined with a differential refractive index detector (RID), is the usual method to track monomer conversion, but when it comes to copolymerization of monomers with similar UV spectra, it does not suffice for the total decoupling of their signals. Here, Fourier‐transform infrared spectroscopy (FTIR) was coupled to an ACOMP system to separate comonomer conversion while monitoring solution copolymerizations of tert‐butyl acrylate (tBA) and n‐butyl acrylate (nBA). The UV spectra of these comonomers are too similar to allow for separation of their conversions during copolymerizaton. The FTIR region between 1100 and 1410 cm−1, however, shows a strong difference between tBA and nBA peaks – 1403 and 1192 cm−1, respectively. FTIR and UV time dependence for the homopolymerization reactions of each monomer were compared, in order to establish that the FTIR is monitoring the same conversion rates. The reaction rates were determined from the FTIR data and that of tBA was higher than that of nBA. As an initial assessment, the terpolymerization of tBA, nBA, and methyl methacrylate (MMA) was also conducted. Reaction rates were found to be significantly lower, and the 1325 cm−1 peak was successfully used for MMA alone. The incorporation of FTIR into ACOMP opens the path for further expansion to a wide variety of copolymerization reactions, including systems of three or more comonomers. It can be a key new detector in the next steps for development of fully automatic feedback control of composition and molecular weight. This article is protected by copyright. All rights reserved

Dynamic light scattering (DLS) is widely used for analyzing biological polymers and colloids. Its application to nanoparticles in medicine is becoming increasingly important with the recent emergence of prominent lipid nanoparticle-(LNP)based products, such as the SARS-CoV-2 vaccines from Pfizer, Inc.-BioNTech (BNT162b2) and Moderna, Inc. (mRNA-1273). DLS plays an important role in the characterization and quality control of nanoparticle-based therapeutics and vaccines. However, most DLS instruments have a single detection angle ,and the amplitude of the scattering vector, q, varies among them according to the relationship q=(n/sin(/2) where 0 is the laser wavelength. Results for identical, polydisperse samples among instruments of varying q yield different hydrodynamic diameters, because, as particles become larger they scatter less light at higher angles, so that higher-q instruments will under-sample large particles in polydisperse populations, and report higher z-average diffusion coefficients, and hence smaller effective hydrodynamic diameters than lower-q instruments. As particle size reaches the Mie regime the scattering envelope manifests angular maxima and minima, and the monotonic decrease of average size versus q is lost. This work examines results for different q-value instruments, using mixtures of monodisperse latex sphere standards, for which experimental measurements agree well with computations, and also polydisperse solutions of LNP, for which results follow expected trends. Mie effects on broad unimodal populations are also considered. There is no way to predict results between two instruments with different q for samples of unknown particle size distributions.

Free radical (FR) and Reversible Addition Fragmentation Chain Transfer (RAFT) copolymerization rates for styrenic monomers in bulk and aqueous reactions were accelerated by acrylic comonomers, whose own rates were both delayed and decreased. The effect was found with styrenics as low as 0.6% by mole, and is directly observable from Automatic Continuous Online Monitoring of Polymerization reactions (ACOMP) data. Rapid radical transfer from acrylic to styrenic comonomer, through lower energy radical resonance stabilization in styrenics, was the suspected cause. FR and RAFT rates were equal, suggesting that FR termination controlled radical concentrations and rates. Despite strongly changing concentrations of initiator and comonomer radicals during reactions, the Quasi-Steady-State Approximation was demonstrated to hold. While rates were identical, molecular weights showed the characteristic increase with conversion for RAFT and decrease for FR. A new, generalized method for reactivity ratio determination was found and acceleration/deceleration behavior was consistently interpreted within a penultimate model.

Although the non-covalent interactions between proteins and salts contributing to the Hofmeister effects have been generally mapped, there are many questions regarding the specifics of these interactions. We report here studies involving the small protein ubiquitin and salts of polarizable anions. These studies reveal a complex interplay between the reverse Hofmeister effect at low pH, the salting-in Hofmeister effect at higher pH, and six anion binding sites in ubiquitin at the root of these phenomena. These sites are all located at protuberances of preorganized secondary structure, and although stronger at low pH, are still apparent when ubiquitin possesses no net charge. These results demonstrate the traceability of these Hofmeister phenomena and suggest new strategies for understanding the supramolecular properties of proteins.

Smart manufacturing will have an enormous effect on the efficiency, economics, and quality of polymeric materials. Very few polymer manufacturing processes are fully optimized, and many follow legacy empirical processes known to yield products with acceptable properties. Optimization of these processes will lead to more efficient use of primarily fossil fuel–based energy and feed stocks, plant assets, and labor, as well as reduced emissions per ton of product; to greater worker and environmental safety by eliminating manual reactor sampling; and to an increase in product consistency and quality. This chapter focuses on the potential for smart polymer manufacturing that is currently developing, based on the automatic continuous online monitoring of polymerization (ACOMP) reaction platform. ACOMP continuously withdraws a minor volume of reactor contents from the process, dilutes, and conditions it as needed, producing a stream of dilute, analytical grade polymer solution, which can then flow through any desired train of detectors to obtain such characteristics as weight average molar mass (Mw), reduced viscosity (RV), conversion rates, and copolymer composition.

Automatic continuous online monitoring and control of polymerization (ACOMP) is a widely applicable platform for monitoring and controlling polymerization reactions. It relies on the continuous extraction, dilution, and conditioning of a small sample stream from the reactor on which measurements by various combinations of detectors are made. By combining simultaneous data from multiple detectors continuous monitoring of salient reaction characteristics can be made, such as kinetics, conversion of comonomers, composition drift, evolution of molecular mass and intrinsic viscosity, and detection of unusual phenomena, such as microgelation and runaway reactions. A growing area for ACOMP is its integration into predictive and closed‐loop reaction control with intriguing possibilities for enhancement by machine learning and artificial intelligence. Typical detectors include light scattering (LS), ultraviolet (UV)/visible spectrophotometry, viscometry, refractivity, and polarimetry, while NMR, dynamic light scattering (DLS), Mie scattering, conductivity, and near infrared (IR) and Fourier‐transform infrared (FTIR) are also used. Recent additions to ACOMP include the ability to use multiple similar sensors to detect the onset and evolution of polymer stimuli responsive behavior during synthesis; for example copolymer compositions at which a lower critical solution temperature (LCST) occurs. ACOMP has been applied to free radical, controlled free radical, and condensation reactions, to micellar, emulsion, and inverse emulsion polymerization, to post‐polymerization and derivatization reactions, and to batch, semi‐batch, and continuous reactors. While ACOMP is not inherently a chromatographic method, its continuous stream can be used in automatic conjunction with gel permeation and other separation chromatographies. Recently, ACOMP has been deployed in full‐scale industrial polymerization reactors to optimize efficiency and product quality.