The utility of employing solid-state NMR (SSNMR) to assess parameters governing the stability of a lyophilized IgG2 protein was the focus of the present work. Specifically, the interaction between the sugar stabilizer (sucrose) and protein component was measured using SSNMR and compared to physical and chemical stability data obtained from thermally stressed samples. 1H T1 and 1H T1⍴ relaxation times were measured by SSMNR for 5 different formulation conditions, and the resultant values were used to examine local mobility and phase separation, respectively. From the SSNMR measurements, it was found local mobility decreased as the sucrose to protein weight ratio increased. The decrease in local mobility corresponded to an increase in storage stability (both chemical and physical) of the lyophilized solids up to a critical weight ratio of sucrose to protein. Additionally, 1H T1⍴ measurements obtained on formulations having higher protein to sucrose weight ratios indicated phase separation of the protein and sucrose phases was occurring, at least on a small scale. Along with an increase in local mobility, phase separation in these specific formulations is thought to have played a role in their decreased storage stability in the solid state.

Background: Surfactant protein-S (SP-D) is a naturally occurring lung protein with the potential to treat pulmonary infections. A recombinant surfactant protein-D (SP-D) has been produced and was previously found to exist in multiple oligomeric states. Introduction: Separation and characterization of interconverting oligomeric states of a protein can be difficult using chromatographic methods, so an alternative separation technique was employed for SPD to characterize the different association states that exist. Methods: Samples of SP-D were analyzed using asymmetrical flow field-flow fractionation (AF4) using UV and multi-angle laser light scattering (MALLS) detection. The AF4 method appears to be able to separate species as small as the monomer up to the dodecamer (the dominant species) to much larger species with a molar mass greater than 5 MDa. Results: Consistent elution of four distinct peaks was observed after repeated injections. The largest species observed under the last peak (labeled as Peak 4) were termed “unstructured multimers” and were resolved fairly well from the other species. The AF4-MALLS data suggest that only a small fraction of Peak 4 truly corresponds to high molar mass unstructured multimers. All other peaks demonstrated significant molar mass homogeneity consistent with AFM results. Conclusion: AF4-MALLS technology appears to be a powerful analytical approach to characterize the complex and dynamic interplay among different protein oligomeric species of SP-D in an aqueous solution.

While asymmetrical flow field-flow fractionation (AF4) has been widely used for separation of high molecular weight species and even particles, its ability to resolve lower molecular weight species has rarely been explored. Over the course of many projects, we have discovered that AF4 can be an effective analytical method for separating peptides from oligomers and higher molecular weight aggregates. The methodology can be used even for peptides as small as 2 kD in molecular weight. Using multi-angle laser light scattering (MALLS) detection, accurate masses of the parent peptide can be obtained, provided accurate extinction coefficients are provided. It was shown that AF4 can be stability-indicating, suggesting that AF4-MALLS may be a suitable alternative to the use of SEC to monitor the aggregation of peptides.

To evaluate the different degrees of residual structure in the unfolded state of interferon-τ using chemical denaturation as a function of temperature by both urea and guanidinium hydrochloride.

Twenty years ago, a number of eminent pharmaceutical scientists collaborated on an article describing a rational approach to developing stable lyophilized protein formulations (Carpenter, Pikal, Chang, & Randolph, 1997). Since that time, no corresponding document for rational development of liquid formulations of proteins has appeared. Certainly, many of the principles underpinning rational protein formulation have been known for some time, but no overarching scheme has ever been described in the literature. Now the time has come to provide a framework for the rational design of protein formulations as aqueous solutions. The objective of this review is to lay out four concepts that will guide one to obtaining a stable liquid protein formulation. Additionally, the aim will be to identify factors that are intrinsic to the stabilization of any protein, not just a particular class of proteins, such as monoclonal antibodies (Uchiyama, 2014; Wang, Singh, Zeng, King, & Nema, 2007) and to provide guidelines aiming to effect stabilization. Noting that all approaches to stabilization face validation that must be performed empirically, it is hoped that the rational strategies described here will help the formulation scientist in their daily tasks and inspire continued advancement of the science involved in protein formulation.

Buffers comprise an integral component of protein formulations. Not only do they function to regulate shifts in pH, they also can stabilize proteins by a variety of mechanisms. The ability of buffers to stabilize therapeutic proteins whether in liquid formulations, frozen solutions, or the solid state is highlighted in this review. Addition of buffers can result in increased conformational stability of proteins, whether by ligand binding or by an excluded solute mechanism. In addition, they can alter the colloidal stability of proteins and modulate interfacial damage. Buffers can also lead to destabilization of proteins, and the stability of buffers themselves is presented. Furthermore, the potential safety and toxicity issues of buffers are discussed, with a special emphasis on the influence of buffers on the perceived pain upon injection. Finally, the interaction of buffers with other excipients is examined.

Determination of the:soluble aggregate levels in protein pharmaceuticals has historically relied upon size-exclusion chromatography (SEC). In recent years, however, there has been a realization that SEC can provide incorrect estimates of the actual aggregate content for a variety of reasons. As a result, orthogonal methods have been evaluated as alternatives to SEC for estimating the amounts and sizes of aggregates in protein products. Of these orthogonal methods, two have been preferred, both in the literature and by regulatory agencies: sedimentation velocity analytical ultracentrifugation (SV-AUC) and field-flow fractionation (FFF), especially asymmetrical-flow field-flow fractionation (AF4). This review describes the relative disadvantages and advantages of these various methods. In doing so, the use of orthogonal methods to SEC is discussed and examples are presented showing how analytical ultracentrifugation (AUC), AF4, and SEC compare in aggregate quantitation.

There is increasing interest in using microalgae as a lipid feedstock for the production of biofuels. Lipids used for these purposes are triacylglycerols that can be converted to fatty acid methyl esters (biodiesel) or decarboxylated to "green diesel." Lipid accumulation in most microalgal species is dependent on environmental stress and culturing conditions, and these conditions are currently optimized using slow, labor-intensive screening processes. Increasing the screening throughput would help reduce the development cost and time to commercial production. Here, we demonstrated an initial step towards this goal in the development of a glass/poly(dimethylsiloxane) (PDMS) microfluidic device capable of screening microalgal culturing and stress conditions. The device contained power-free valves to isolate microalgae in a microfluidic growth chamber for culturing and stress experiments. Initial experiments involved determining the biocompatibility and culturing capability of the device using the microalga Tetraselmis chuii. With this device, T. chuii could be successfully cultured for up to 3 weeks on-chip. Following these experiments, the device was used to investigate lipid accumulation in the microalga Neochloris oleabundans. It was shown that this microalga could be stressed to accumulate cytosolic lipids in a microfluidic environment, as evidenced with fluorescence lipid staining. This work represents the first example of microalgal culturing in a microfluidic device and signifies an important expansion of microfluidics into the biofuels research arena.

Metabolomics is an emerging area of research focused on measuring small molecules in biological samples. There are a number of different types of metabolomics, ranging from global profiling of all metabolites in a single sample to measurement of a selected group of analytes. Microfluidics and related technologies have been used in this research area with good success. The aim of this review article is to summarize the use of microfluidics in metabolomics. Direct application of microfluidics to the determination of small molecules is covered first. Next, important sample preparation methods developed for microfluidics and applicable to metabolomics are covered. Finally, a summary of metabolomic work as it relates to analysis of cellular events using microfluidics is covered.

Selectivity and resolution for analyses conducted using microfluidic devices can be improved by increasing the total number of individual detection elements in the device. Here, a poly(dimethylsiloxane) capillary electrophoresis microchip was fabricated with an integrated electrode array for selective detection of small molecules. Eight individually addressable gold electrodes were incorporated in series after a palladium current decoupler in the separation channel of an electrophoresis microchip. The electrode array device was characterized using a mixture of biologically relevant analytes and xenobiotics: norepinephrine, 4-aminophenol, acetaminophen, uric acid, and 3,4-dihydroxyphenylacetic acid. Separation efficiencies as high as 9000 +/- 1000 plates (n = 3) for 3,4-dihydroxyphenylacetic acid and limits of detection as low as 2.6 +/- 1.2 microM (n = 3) for norepinephrine were obtained using this device. After characterizing the performance of the device, potential step detection was conducted at the array electrodes and selective detection achieved based upon differences in redox potentials for individual analytes. Utilization of potential step detection was particularly advantageous for resolving co-migrating species; resolution of 3,4-dihydroxy-l-phenylalanine from acetaminophen using potential control was demonstrated. Finally, a human urine sample was analyzed using potential step detection to demonstrate the applicability of this device for complex sample analysis.