Reduced properties, also known as amino acid descriptors, quantify changes in physicochemical properties of amino acids upon mutation. Combined with PLS (partial least squares) modeling, the impact of various mutations on the conformational stability of cold shock proteins (CSPs) was examined. Consistent with the initial evaluation of these systems, electrostatic properties at a select number of central residues were found to govern the conformational stability of CSP mutants. In addition, the current studies found that packing efficiency within the β-sheet core and flexibility of the peptide backbone also contribute to the overall stability of these small proteins.

A chemometric approach using amino acid descriptors was employed to evaluate the effect of various mutations on the aggregation propensity of human calcitonin (hCt), an approved peptide therapeutic. Models based on well-established amino acid descriptors, like z-scores and F scores, explained nearly all of the variance in aggregation propensity. The likelihood of a mutant to aggregate was found to depend largely on hydrophobicity of key residues, although interactions between aromatic residues are also important. Residues within the central core of the peptide were influential, along with specific sites in the C-terminal region. Meanwhile, electrostatic interactions play only a minor role in governing hCt aggregation. In contrast to previous studies, Phe19 and Tyr12 were found not to have a significant impact on aggregation propensity, whereas Phe16 and Phe22 exerted a somewhat larger effect. The importance of specific C-terminal residues on aggregation propensity of hCt has now been delineated. These findings are relevant to the manufacturing and handling of hCt and are also of importance to its clinical utilization. This work could also guide the potential development of new biobetters of human calcitonin.

A novel neurotrophic factor, human mesencephalic astrocyte-derived neurotrophic factor (hMANF), is being considered a therapeutic agent for a variety of diseases. However, little, if anything, has been reported about its stability. A preformulation study was conducted to assess the stability of hMANF as a function of pH and temperature. In addition, the effects of buffers and other excipients were evaluated as well. While the chemical and physical stability of hMANF decreases near pH 4, overall, the protein appears to be quite stable, especially near pH 6. Both histidine and phosphate appear to be suitable buffers in this pH range. Some loss of stability was noted above pH 6.5 as well. The stability profile of hMANF was comparable at 1 and 10 mg/mL. The decreased stability at acidic pH is correlated with the loss of the native α-helical conformation, as shown by FTIR spectroscopy. These studies indicate that hMANF is quite stable near pH 6, and formulations capable of exhibiting adequate long-term stability in aqueous solutions should be possible.

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.

The field of formulation and stabilization of protein therapeutics has become rather extensive. However, most of the focus has been on stabilization of the final drug product. Yet, proteins experience stress and degradation through the manufacturing process, starting with fermentaition. This review describes how formulation principles can be applied to stabilize biopharmaceutical proteins during bioprocessing and manufacturing, considering each unit operation involved in prepration of the drug substance. In addition, the impact of the container on stabilty is discussed as well.

Background: The importance of aromaticity vs. hydrophobicity of the central hydrophobic core (CHC, residues 17-20) in governing fibril formation in A beta(1-42) has been the focus of an ongoing debate in the literature. Introduction: Mutations in the CHC (especially at Phe19 and Phe20) have been used to examine the relative impact of hydrophobicity and aromaticity on the degree of aggregation of A beta(1-42). However, the results have not been conclusive. Methods: Partial least squares (PLS) modeling of aggregation rates, using reduced properties of a selies of position 19 mutants, was employed to identify the physicochemical properties that had the greatest impact on the extent of aggregation. Results: The PLS models indicate that hydrophobicity at position 19 of A beta(1-42) appears to be the primary and dominant factor in controlling A beta(1-42) aggregation, with aromaticity having little effect. Conclusion: This study illustrates the value of using reduced properties of amino acids in conjunction with PLS modeling to investigate mutational effects in peptides and proteins, as the reduced properties can capture in a quantitative manner the different physicochemical properties of the amino acid side chains. In this particular study, hydrophobicity at position 19 was determined to be the dominant property controlling aggregation, while size, charge, and aromaticity had little impact.

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.

Protein aggregates and subvisible particles (SbvP), inherently present in all marketed protein drug products, have received increasing attention by health authorities. Dynamic imaging analysis was introduced to visualize SbvP and facilitate understanding of their origin. The educational United States Pharmacopeia chapter <1787> emphasizes that dynamic imaging analysis could be used for morphology measurements in the size range of 4-100 μm. However, adequate morphology characterization, as suggested in the United States Pharmacopeia <1787> proposed size range, remains challenging as nonspherical size standards are not commercially available. In this study, a homogenous and well-defined nonspherical particle standard was fabricated and used to investigate the capabilities of 2 dynamic imaging analysis systems (microflow imaging (MFI) and FlowCAM) to characterize SbvP shape in the size range of 2-10 μm. The actual aspect ratio of the SbvP was measured by scanning electron microscopy and compared to the results obtained by dynamic imaging analysis. The test procedure was used to assess the accuracy in determining the shape characteristics of the nonspherical particles. In general, dynamic imaging analysis showed decreasing accuracy in morphology characterization for 5 μm and 2 μm particles. The test procedure was also capable to compare and evaluate differences between the 2 dynamic imaging methods. The present study should help to define ranges of operation for dynamic imaging analysis systems.

A number of algorithms have been developed to predict the aggregation propensity of peptides and proteins, but virtually none have the ability to provide sequence-specific information on what physicochemical properties are most important in altering aggregation propensity. In this study, a chemometric approach using reduced amino acid properties is used to examine the aggregation behavior of a highly amyloidogenic peptide, Aβ(1-42). Specific residues are identified as being critical to the aggregation process. At each of these positions, the important physicochemical properties are identified that would either accelerate or inhibit fibril formation.

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.

Deamidation of asparagine (Asn) residues is one of the most common chemical degradation pathways observed in proteins. This reaction must be understood and controlled in therapeutic drug candidates, as chemical changes can affect their efficacy and safety. The analytical tools available for detection of deamidation reaction products, such as isoaspartic acid residues, are either chromatographic or electrophoretic, and require MS detection for absolute identification of peaks. High-throughput measurement of protein degradation has typically been limited to probing the target's physical state using spectroscopic techniques. Here, we describe a high throughput assay for isoaspartate residues using fluorescent detection in a microtiter plate format. The method allows for fast detection of protein deamidation in a cost-efficient manner. The method can be employed even if the target peptide or protein contains free Cys residues. The technique appears to be selective, linear, and accurate.

The peptide teriparatide, also known as parathyroid hormone (1-34), PTH(1-34), was developed for intranasal delivery, requiring extended stability of the reconstituted product for up to four weeks at room temperature. Lyophilized formulations of PTH(1-34), containing glycine and trehalose and using lactate as the buffer, are stable for months upon storage. However, the physical stability of the peptide after reconstitution unexpectedly varied considerably, depending on peptide concentration and storage temperature, with precipitation seen within two to four weeks in some samples. By comparison, equivalent samples that did not undergo lyophilization did not display any precipitation upon storage in the liquid state for as long as twelve weeks. PTH(1-34) appears to adopt a higher order structure that is perturbed by the combined stresses of freezing and drying, leading to greater propensity to aggregate, which is accentuated at higher peptide concentrations and at higher temperatures. The precipitation seems to be correlated with increased amounts of subvisible particles. This study shows the importance of peptide conformation in long-term stability and illustrates the ability of lyophilization to cause increased propensity to aggregate, even in a peptide.

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.